1521-009X/41/3/634–641$25.00
DRUG METABOLISM AND DISPOSITION
Copyright ª 2013 by The American Society for Pharmacology and Experimental Therapeutics
http://dx.doi.org/10.1124/dmd.112.048983
Drug Metab Dispos 41:634–641, March 2013
Amide Hydrolysis of a Novel Chemical Series
of Microsomal Prostaglandin E Synthase-1 Inhibitors
Induces Kidney Toxicity in the Rat
Johan Bylund, Anita Annas, Dennis Hellgren, Sivert Bjurström, Håkan Andersson,
and Alexander Svanhagen
Drug Metabolism and Pharmacokinetics, CNSP iMed Science (J.B.), and Safety Assessment (A.A., D.H., S.B., H.A., A.S.),
AstraZeneca R&D, Innovative Medicines, Södertälje, Sweden; and Department of Pharmaceutical Biosciences, Uppsala Biomedical
Centre, Uppsala University, Uppsala, Sweden (J.B.)
Received September 10, 2012; accepted January 2, 2013
A novel microsomal prostaglandin E synthase 1 (mPGES-1) inhibitor
induced kidney injury at exposures representing less than 4 times the
anticipated efficacious exposure in man during a 7-day toxicity study
in rats. The findings consisted mainly of tubular lesions and the
presence of crystalline material and increases in plasma urea and
creatinine. In vitro and in vivo metabolic profiling generated a working
hypothesis that a bis-sulfonamide metabolite (determined M1)
formed by amide hydrolysis caused this toxicity. To test this hypothesis, rats were subjected to a 7-day study and were administered
the suspected metabolite and two low-potency mPGES-1 inhibitor
analogs, where amide hydrolysis was undetectable in rat hepatocyte
experiments. The results suggested that compounds with a reduced
propensity to undergo amide hydrolysis, thus having less ability to
form M1, reduced the risk of inducing kidney toxicity. Rats treated
with M1 alone showed no histopathologic change in the kidney, which
was likely related to underexposure to M1. To circumvent rat kidney
toxicity, we identified a potent mPGES-1 inhibitor with a low
propensity for amide hydrolysis and superior rat pharmacokinetic
properties. A subsequent 14-day rat toxicity study showed that this
compound was associated with kidney toxicity at 42, but not 21,
times the anticipated efficacious exposure in humans. In conclusion,
by including metabolic profiling and exploratory rat toxicity studies,
a new and active mPGES-1 inhibitor with improved margins to
chemically induced kidney toxicity in rats has been identified.
Introduction
derivatives, has two acidic pKa values around 2.5 and 10.5, CLogP
values exceeding 2.5 and molecular weight exceeding 430 Da.
Considering their molecular size and lipohilicity, parent compounds of
this series and their conjugated metabolites are likely excreted into the
bile (Yang et al., 2009), which differs from the antibiotic class of
sulfonamides (Fleck et al., 1988; Perazella, 1999).
By assessing possible in vivo safety concerns in relation to anticipated efficacious exposures in humans already within the discovery
program, drug candidates with a better chance of success in preclinical
testing can be brought forward. mPGES-1 is an enzymatic drug target
involved in the production of PGE2 (prostaglandin E2), a mediator
of pain and inflammation (Samuelsson et al., 2007). It has been
hypothesized that mPGES-1 inhibition might generate pain relieving
anti-inflammatory effects similar to cyclooxygenase (COX) inhibitors
such as conventional NSAIDs (nonsteroidal anti-inflammatory drugs)
and COX-2 inhibitors (Samuelsson et al., 2007; Xu et al., 2008; Iyer
et al., 2009). However, mPGES-1 inhibitors differ from COX inhibitors
with respect to where in the biosynthetic cascade they inhibit PGE2
production. It has been hypothesized that mPGES-1 inhibitors might
exhibit better safety profiles due to their propensity to affect the
formation of other eicosanoids than PGE2 in a different way than
conventional NSAIDs and COX-2 inhibitors (Samuelsson et al.,
2007; Wang and FitzGerald 2010). However, this might also have
Deposition of drug-induced crystals within the kidneys can lead to
acute and chronic kidney injury due to obstruction (Perazella, 1999;
Yarlagadda and Perazella, 2008). A variety of drugs have been shown
to precipitate in the renal tubules as a consequence of high local
concentrations in the kidney and changes in drug solubility due to pH
differences between urine and blood. One group of drugs that has been
shown to cause kidney injury by crystalluria is the bacteriostatic
antibiotic class of sulfonamides (Perazella, 1999). The sulfonamide
drugs such as sulfadiazine and sulfamethoxazole are to a large extent
excreted in the urine as parent molecules or metabolites. Because they
are weak acids, they are relatively insoluble in acidic urine and have
a tendency to precipitate in the tubular lumen when the pH of the urine
drops below 5.5 (Hein et al., 1993; Perazella, 1999). High-dose
sulfonamide treatment has been associated with an increased risk of
sulfa-crystal precipitation in the kidneys (Perazella, 1999).
A new chemical series of sulfonamide-containing compounds was
recently identified as potential microsomal prostaglandin E2 synthase1 (mPGES-1) inhibitors intended for pain treatment (Bylund et al.,
2009). This series, which is composed of different bis-sulfonylamino
dx.doi.org/10.1124/dmd.112.048983.
ABBREVIATIONS: AUC0–24, area under the curve for 24 hours; AUCther, area under plasma concentration time curve needed for therapeutic effect;
COX, cyclooxygenase; LC-MS, liquid chromatography mass spectrometry; LC-MS/MS, liquid chromatography-tandem mass spectrometry;
mPGES-1, microsomal prostaglandin E synthase-1; NSAID, nonsteroidal anti-inflammatory drug; PGE2, prostaglandin E2; PK, pharmacokinetics;
TK, toxicokinetics; WBA, whole blood assay.
634
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ABSTRACT
Amide Hydrolysis-Induced Kidney Toxicity
Materials and Methods
Chemicals and Reagents. Test compounds, including the metabolites referred to as M1 and Hyd, originated from AstraZeneca’s compound collection
(Södertälje, Sweden), and the synthesis of such derivatives has been described
in a patent (Bylund et al., 2009). Chemical structures of AZ’7847, AZ’4270,
AZ’4284, AZ’0908, and M1 are shown in Fig. 1. Those labeled [14C]-AZ’7847
and [14C]-AZ’4270 were synthesized by the isotope chemistry laboratory at
AstraZeneca, Södertälje, Sweden, with a specific activity of 2.1 GBq/mmol and
a radiochemical purity exceeding 99.4%. The 14C label was located, for both
compounds, on the carboxyl carbon as depicted in Fig. 5. All other chemicals
used were of analytical grade and obtained from commercial suppliers.
Solubility Measurements and pKa Predictions. Solubility samples in rat
urine (pH 7.0) were incubated at 37°C for 5 hours and analyzed using a Hewlett
Packard 1100 system (Agilent Technologies, Santa Clara, CA) equipped with
a Symmetry C18 column (3.5 mm, 4.6 50 mm), a photodiode array detector,
and a mass single detector. The mobile phases consisted of water with 0.3%
trifluoroacetic acid (phase A) and acetonitrile with 0.3% trifluoroacetic acid
(phase B). A 7-minute-long gradient starting at 50% and ending at 5% phase A
was used at a flow rate of 0.5 ml/min. Detection was performed at 280 nm, 305
Fig. 1. Structural formula of compounds AZ’7847, M1, AZ’4270, AZ’4284, and
AZ’0908 discussed in the text.
nm, and 335 nm. Predictions of pKa were generated by an in-house model built
from AstraZeneca’s internal data sets and physicochemical descriptors.
In Vitro Metabolic Experiments. Fresh and cryopreserved human
hepatocytes were supplied by CellzDirect, Inc. (Durham, NC) and were handled
as previously described elsewhere (Floby et al., 2009; Sohlenius-Sternbeck et al.,
2010). The cryopreserved human hepatocytes were composed of a mix of
hepatocytes isolated from several male and female subjects, whereas the fresh
human hepatocytes were isolated from one male (Hu8001) and one female
(Hu0587) single donors. Fresh rat hepatocytes were isolated from male Sprague
Dawley rats, as previously described elsewhere (Floby et al., 2009; SohleniusSternbeck et al., 2010). Cryopreserved male Beagle dog hepatocytes were obtained
from XenoTech (Lenexa, KS). Hepatocyte incubations were conducted as
previously described elsewhere (Sohlenius-Sternbeck et al., 2010). Briefly,
hepatocytes at concentrations 1 106 cells/ml in William’s medium were
incubated at 37°C with gentle shaking in a 5% CO2 atmosphere at substrate
concentrations of either 1 mM or 10 mM. All incubations were stopped by the
addition of 2 volumes of ice-cold acetonitrile and were then centrifuged
before liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.
The metabolic capacity of hepatocytes was checked in-house using a set of probe
substrates (Floby et al., 2009).
Liquid Chromatography Mass Spectrometry Analysis. Liquid chromatography mass spectrometry (LC-MS) analysis for quantifications was performed
with a Micromass Quattro Micro triple quadrupole (Waters Corporation, Milford,
MA), as previously described elsewhere (Briem et al., 2007) with the exception of
analysis of the M1 metabolite. Total concentration of M1 in plasma was
determined by LC-MS/MS using electrospray in the negative ionization mode.
Because the analyte was not retained on a conventional C18 column, a porous
graphitic carbon column and methanol gradient were used. For quantification of
the M1 metabolite, the mass spectrometer was set to select the [M-H]2 precursor
ion at mass-to-charge ratio 234.9 and for the product ion at mass-to-charge ratio
171.1. Before the LC-MS/MS analysis, the samples were precipitated with
acetonitrile.
The LC-MS analysis for metabolic profiling was performed as described
here. Liquid chromatography was performed with Schmidazu LC-10AD VP
pumps (Schimadzu Deutschland, Duisburg, Germany) and a HTC PAL (CTC
Analytics AG, Zwingen, Switzerland). The separations were performed on
a Waters Atlantis T3 column (100 2.1 mm ID, 3 mm particle size) at a flow
rate of 0.25 ml/min. The mobile phase was a binary mixture of 0.1% formic
acid in water/acetonitrile (98:2), v/v (solvent A), and 0.1% formic acid in water/
acetonitrile (20:80), v/v, (solvent B). Mass spectrometry was performed on an
LTQ Orbitrap hybrid Fourier transform mass spectrometer (Thermo Scientific,
Waltham, MA). An electrospray interface in the negative ion mode was used in
all experiments. The following scan events were used: event 1: survey scan
120–900 amu; event 2: data-dependent scan. Product ion scans comprised of
the most intense ion from scan event 1. The software Xcalibur 2.0 (Thermo
Scientific) was used for data acquisition, processing, and control of the mass
spectrometer. MetWorks version 1.2.0 (Thermo Scientific) was used for
tentative identification of metabolites with accurate mass using a mass tolerance
value of 30 ppm. The separations for radio detection were performed on
a Waters Atlantis T3 column (100 4.6 mm ID, 3 mm particle size). The flow
rate was 1 ml/min with a 1:20 split to the ion source and flow scintillation
monitor (625 TR; PerkinElmer Life and Analytical Sciences, Boston, MA).
Scintillation cocktail (Ultima Flo-M; PerkinElmer Life and Analytical
Sciences) was pumped at a flow rate of 4 ml/min to the flow scintillation
analyzer, which was equipped with a 0.5 ml flow cell.
Animals. Wistar Hannover rats, approximately 8 weeks old, were used for
toxicity studies, and male Sprague-Dawley rats were used for pharmacokinetic
(PK) studies. All rats were obtained from Taconic M&B A/S (Ry, Denmark).
The rats were randomized into groups (n = 2–4/sex) by body weight and were
acclimatized for approximately 2 weeks before the start of the study. The
animals were housed up to 3 per cage in transparent plastic cages. The bedding
material consisted of aspen wood chips. Plastic tunnels and aspen chew blocks
were also provided. The animals had free access to pelleted RM1 (E) SQC diet
and to water from the site drinking water supply, except during urine collection
when access to food was temporarily withdrawn. The animal room was
illuminated by artificial light from fluorescent tubes on a 12-hour light/dark
cycle. The temperature and relative humidity were 17° to 23°C and 40 to 70%,
respectively. All experiments were approved by the local animal ethics
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consequences for the translational aspects of PGE2 inhibition in pain
relief (Wang and FitzGerald 2010).
NSAIDs and COX-2 inhibitors have previously shown good correlation between the average steady-state exposure in human subjects
with a favorable analgesic effect and approximately 80% inhibition
in a well established whole blood assay (WBA) that measures
lipopolysaccharide-induced COX-2-mediated production of the biomarker PGE2 (Depré et al., 2000; Huntjens et al., 2005; Huntjens
et al., 2006). The criterion for efficacious therapeutic concentration
for this mPGES-1 inhibition project was based on an assumption that
a steady-state concentration average corresponding to 10 times the
WBA IC50 value would be satisfactory for pain treatment. However,
it is presently unknown whether mPGES-1 inhibitors are efficacious
for pain treatment in humans and whether the WBA can be used as
a translational tool for this novel group of drugs.
In an attempt to develop a novel mPGES-1 inhibitor, we experienced problems associated with chemically induced kidney
toxicity in the rat at exposures representing less than 4 times the
anticipated efficacious exposure in man. The aim of this study is to
describe problem-solving strategies in a drug discovery project
encumbered with kidney toxicity. More specifically, we describe the
toxicologic kidney findings observed in a 7-day toxicity study,
the establishment of a hypothesis for toxicity origination, and the
discovery of a new inhibitor with a reduced kidney toxicity risk
profile.
635
636
Bylund et al.
TABLE 1
Toxicity study design
Tox Study
Test Duration
Test Compound
1
7
2
7
Vehicle
AZ’7847
Vehicle 1a
Vehicle 2b
AZ’7847
Ml
AZ’4270
AZ’4284
3
14
Dose
Sex and No. of Animals
Comments on TK Samples
mmol/kg
days
AZ’0908
0
500
1000
0
0
1000
500
2000
170
500
76
230
435
900
2M + 2F
2M + 2F
2M + 2F
4F
4F
4F
4F
4F
4F
4F
4F
4F
10F
10F
2M + 2F satellite animals
2M + 2F satellite animals
2F
2F
2F
2F
2F
2F
2F
5F
5F
satellite
satellite
satellite
satellite
satellite
satellite
satellite
animals
animals
animals
animals
animals
animals
animals
animals
animals
for m-sampling
for m-sampling
committee (Stockholms Södra försöksdjursetiska nämnd) and were conducted
in compliance with national guidelines for the care and use of laboratory
animals.
Toxicology Studies. Three different rat toxicologic studies were performed.
A detailed description of the study design is shown in Table 1. Compounds
AZ’7847, AZ’4270, and AZ’4284 were each formulated as solutions in 20%
(v/v) polyethylene glycol 400 in 0.2 M meglumine (vehicle 1); the M1
metabolite was formulated a solution in 1% (w/v) hydroxypropyl methylcellulose and 0.1% (w/v) Tween 80 (vehicle 2); and AZ’0908 was formulated as
solution in 15% (w/v) hydroxypropyl-b-cyclodextrin in 0.2 M meglumine
(vehicle 3). Animals used for toxicity studies were dosed once daily by oral
gavage (10 ml/kg body weight) for 7 or 14 days. Control animals were treated
with either vehicle 1 or vehicle 2. Doses were set based on low-dose PK studies
where linear kinetics was assumed.
Toxicokinetics. Two male and two female satellite rats (animals used for
exposure analysis only) were used for toxicokinetic (TK) analysis per dose
group in the first study, but only 2 satellite female rats were used per dose
group in the second study with AZ’7847 and AZ’4270, and AZ’4284 and the
M1 metabolite. In the third toxicity study, five animals per dose group of
AZ’0908 were used for TK analysis. All compounds were dosed once daily for
7 consecutive days, and blood samples were taken at 2, 4, 6, 7, and 24 hours on
day 1 and on the last day of administration. For satellite animals in toxicity
studies 1 and 2, approximately 0.3 ml of blood was taken via the tail vein and
was collected in EDTA Microtainer tubes (BD, Plymouth, UK). The blood
samples were immediately placed on ice, and they were centrifuged within 30
minutes at +4°C for 10 minutes at 1500g to obtain plasma. The plasma was
transferred to 1.4 ml Matrix vials and was immediately frozen to 270°C. The
samples were analyzed for only the parent molecule in toxicity studies 1 and 3,
but both the parent molecules and M1 metabolite were analyzed in toxicity
study 2. In toxicity study 3, a microsampling technique was used to sample data
for toxicokinetic analysis (Jonsson et al., 2012). The TK parameters Cmax and
area under the curve for 24 hours (AUC0–24) were calculated with Pharsight
WinNonlin (Pharsight, St. Louis, MO). All TK values are presented as mean
values from at least two animals. Low-dose PK studies for dose setting in
toxicity studies were performed as described elsewhere (Briem et al., 2007).
Clinical Pathology. Blood plasma samples (into tubes with lithium heparin)
were taken from the orbital plexus of animals anesthetized with isoflurane and
oxygen at the end of the dosing period; they were analyzed for urea and
creatinine. The detection of urea and creatinine levels was performed on
a clinical chemistry analyzer COBAS 6000 c501 (instrument and reagents by
Roche, Penzberg, Germany). The limit of detection was 0.5 mmol/l and
5 mmol/l, respectively. Individual urine samples from animals placed in
metabolism cages were collected on ice for about 6 hours starting immediately
after dosing and were analyzed for crystals by cytologic examination.
Histopathology. The rats were killed by exsanguination from a common
carotid artery under isoflurane and oxygen anesthesia 1 day after the final dose
was given. The body weight was recorded, and the kidneys were removed,
fixed, and preserved in buffered formalin. The preserved kidney specimens
were trimmed, processed through graded alcohol and clearing agent, infiltrated,
and embedded in paraffin, sectioned (5 mM), and stained with hematoxylin and
eosin (Bancroft and Gamble, 2008). The sections were examined for
histopathologic changes using a light microscope.
Results
Kidney Toxicity of AZ’7847. Estimations of efficacious therapeutic concentration in humans (AUCther) were based on an assumption
that an average steady-state concentration corresponding to 10 times
the attained IC50 value (unpublished data) in a well established whole
blood PGE2 assay (Huntjens et al., 2005) would be satisfactory for
pain treatment. Doses aiming to achieve 10 and 20 times the AUCther
TABLE 2
Toxicokinetic summary of AZ’7847 in toxicity study 1
Day 1
Dose
500
500
1000
1000
(mmol/kg)
(mmol/kg)
(mmol/kg)
(mmol/kg)
Sex and No. of Animals
2M
2F
2M
2F
Day 7
Anticipated AUCther (mMh)a
115
115
115
115
AUC0–24/AUCther (ratio)
Cmax
AUC0–24
Cmax
AUC0-24
mM
mMh
mM
mMh
73
38
46
75
840
450
610
1020
68
39
NA
NA
480
430
NA
NA
4.2
3.7
NA
NA
AUC0–24, area under the curve for 24 hours; AUCther, area under plasma concentration time curve needed for therapeutic effect; F, female; M, male; NA, not available.
a
AUCther (area under plasma concentration time curve needed for therapeutic effect) was based on the assumption that a steady-state concentration (Css) average corresponding to 10 times the whole
blood assay IC50 value was needed.
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F, female; M, male; TK, toxicokinetics.
a
Same vehicle as AZ’7847, AZ’4270, and AZ’4284.
b
Same vehicle as for M1.
Amide Hydrolysis-Induced Kidney Toxicity
637
Fig. 2. Histopathologic changes in the rat kidney papilla. (A) Control animal treated with the same formulation as AZ’7847 for the same time period. (B) Distinct kidney
necrosis, inflammation, and crystal deposits were detected in the rats treated with AZ’7847. Kidneys stained with hematoxylin and eosin; magnification, 100.
AZ’7847 per day for 7 days. Analysis of plasma showed extremely
high levels of urea and creatinine in rats treated with 1000 mmol/kg
per day for 7 days (Fig. 3). At the low dose, 500 mmol/kg, moderate
tubular degeneration/regeneration was observed. The characteristic
kidney damage and identification of crystal deposits suggested that
kidney toxicity was caused by tubular precipitation of AZ’7847associated drug material.
Amide Hydrolysis of AZ’7847. Metabolic profiling in rat
hepatocytes showed that amide hydrolysis of AZ’7847 was a major
metabolic pathway in vitro. Comparison of chromatograms detected by
mass spectrometry (Fig. 4A) and radiodetection (Fig. 4B) showed that
the carboxylic acid metabolite formed (depicted as Hyd in Fig. 5) had
a much lower mass spectrometry response than the parent compound,
whereas the corresponding bis-sulfonamide metabolite (depicted as M1
in Fig. 5) was not detected by either MS analysis or radiodetection. That
amide hydrolysis was a major metabolic pathway in vivo was confirmed
by metabolic profiling of the plasma samples from the toxicity study
(Fig. 4C). Additional major metabolites tentatively identified by
accurate mass LC-MS analysis were a mono-oxidized metabolite
(marked +16), a combined oxidized and sulfate-conjugated metabolite
Fig. 3. Kidney functional biomarkers. (A) Urea. (B) Creatinine. Mean values generated from two male and two female rats. The standard error of the mean is shown in
graphs.
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in humans were used. Rats were orally administered either 500 or 1000
mmol/kg per day for 7 consecutive days. LC-MS and TK analyses
revealed that a lower than anticipated exposure on day 7 for low-dose
animals was achieved and that no obvious sex difference in AUC was
detected (Table 2). No high-dose exposure data at day 7 were available
due to premature death or sacrifice.
Toxicologic evaluation showed severe kidney injury in animals from
the high-dose group. Necropsy findings included increased kidney
weights and pale kidney discoloration. The histopathologic evaluation
showed marked tubular degeneration, characterized by the presence of
dilated tubules with cellular remnants within the lumen, mixed with
basophilic, dilated tubules with hypertrophic nuclei and cytoplasm. The
distribution of this finding was cortex and medulla, with emphasis
on the medulla. Additional changes in the kidney included multiple
pyogranuloma (accumulation of neutrophils/macrophages surrounding
amorphous/crystalline material within tubules in the cortex and medulla),
inflammation and hemorrhage in the renal pelvis, free crystals in the renal
pelvis, transitional cell hyperplasia, and papillitis.
Figure 2 shows the histopathology comparison of a kidney from
a control rat and from the kidney of a rat treated with 1000 mmol/kg
638
Bylund et al.
(marked +16+Sulf), a metabolite likely formed by amide hydrolysis in
combination with mono-oxidation (Hyd+16), and a metabolite formed
by amide hydrolysis in combination with mono-oxidation and sulfateconjugation (Hyd+16+Sulf). Metabolic in vitro profiling in both
cyropreserved and freshly prepared human hepatocytes and cryopreserved dog hepatocytes revealed no indication that AZ’7847 had
undergone amide hydrolysis. Metabolic profiling experiments in
human hepatocytes suggested that AZ’7847 is highly metabolically
stable (Fig. 4D).
The detection of large quantities of the Hyd metabolite in rats
obviously suggested formation of large amounts of the corresponding
M1 metabolite (Fig. 5). Measurements of the M1 solubility in rat urine
at pH 7.0 revealed a moderate solubility of 83 mM, which was similar
to the parent molecule (45 mM). The acidic pKa of the M1 metabolite
was predicted to be approximately 9.5. Based on the detection of this
M1 metabolite that structurally resembles sulfonamide-containing
antibacterial agents and the characteristic kidney damage, we hypothesized that a compound with a low degree of hydrolysis would be
superior with respect to kidney toxicity risk.
Evaluation of Kidney Toxicity Hypothesis. Two compounds,
AZ’4270 and AZ’4284 (Fig. 1), containing structural modifications
intended to block amide hydrolysis by steric hindrance were
identified. No hydrolysis in rat hepatocytes (Fig. 6, A and B) was
detected for either of the two compounds, and they were chosen for an
exploratory rat toxicity study. Both compounds exhibited IC50 values
above 2 mM in the human WBA (unpublished data) and were
therefore only considered as tool compounds. A 7-day rat toxicity
study focusing on kidney toxicity and M1 exposure was performed
with the M1 metabolite and the compounds AZ’4270 and AZ’4284
together with AZ’7847 as a positive control (toxicity study 2 in
Table 1). Doses were set to achieve a similar and a significantly higher
exposure than what had been achieved by 1000 mmol/kg AZ’7847 in
the previous study. LC-MS and TK analyses showed that the
exposures of AZ’4270 and AZ’4284 per dose unit were more than 5
times higher than for AZ’7847 (Table 3). TK analysis also showed that
the M1 metabolite in the plasma samples of rats administered
AZ’4270 and AZ’4284 was much lower than with the positive control
AZ’7847. The ratio between the exposure of the M1 metabolite and
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Fig. 4. Metabolic profiling of AZ’7847. (A) Rat hepatocyte metabolites detected with LC-MS. (B) Rat hepatocyte metabolites detected with radio monitoring [same LC
chromatogram as in (A)]. (C) In vivo rat metabolism detected with LC-MS. Plasma sample used for analysis originated from of a low-dose (500 mmol/kg per day) rat at
4 hours on day 7. (D) Freshly prepared human hepatocytes analyzed with LC-MS. The MS chromatograms are showing the combined base peak intensities of the molecular
ion [M-H]2 of the parent molecule m/z 439.042 and the potential metabolites with m/z values 221.060, 237.055, 317.012, 397.092, 453.021, 455.037, 471.032, 473.048,
493.048, 534.994, 631.069, and 762.121. Putative peak identification was suggested by LC-MS analysis with accurate mass determination. The Hyd metabolite identification
was confirmed by comparison of MS/MS spectra and LC retention time generated with a synthesized standard.
Amide Hydrolysis-Induced Kidney Toxicity
the exposure of the parent molecule at day 7 was more than 30 times
lower for AZ’4270 and more than 75 times lower for AZ’4284 as
compared with AZ’7847. The exposure of M1 after M1 administration
was significantly lower than for the positive control AZ’7847, and
approximately the same exposure was achieved after the administration
of 500 and 2000 mmol/kg per day (Table 3). M1 accumulation appeared
to be low during the administration period, except in rats treated with
AZ’7847 in whom both AUC0-24 and Cmax increased more than 3-fold.
The histopathologic analysis of kidneys from rats treated with
AZ’7847 was similar to the previous study, and elevated levels of the
kidney functional biomarkers urea and creatinine were detected (not
shown). Neither pathophysiologic findings nor elevation of kidney
functional biomarkers were detected for AZ’4270 and AZ’4284
despite exposure levels of parent molecules in the same range or
higher than AZ’7847. However, crystals were seen in the urine of
animals treated with AZ’4270 (only in high dose) but not with
AZ’4284. No histopathologic findings or changes in creatinine and
urea parameters were detected for rats treated with the M1 metabolite.
Crystals were observed in the urine from both dose groups of animals
dosed with the M1 metabolite.
Identification and Evaluation of AZ’0908. We identified a novel
and potent mPGES-1 inhibitor AZ’0908 (Fig. 1), which exhibited no
detectable amide hydrolysis in rat hepatocytes (not shown). A rat PK
study using a single dose of 10 mmol/kg (Fig. 7) suggested that the rat
exposure per dose unit of AZ’0908 and the ratio of the plasma
concentration of the M1 metabolite and the parent molecule were
drastically better than for AZ’7847 (Table 4). Considering the improved rat PK and possibly also improved redirection of metabolism,
AZ’0908 was chosen for evaluation in a 14-day rat study focusing on
kidney toxicity (toxicity study 3 in Table 1).
Doses were set to achieve approximately 20-fold and 40-fold
margins to anticipated AUCther in humans. Samples for TK analysis
were taken by microsampling, and the M1 metabolite was not
monitored in the study. TK analysis showed that the exposure
objectives were achieved and that the two doses exhibited doseproportionality for AUC0–24 but not for Cmax (Table 3). Toxicologic
evaluation showed that the low dose, which generated exposures of 21
times the anticipated AUCther in humans, was not associated with any
significant kidney toxicology. However, for 6 out of 10 animals in the
high-dose group tubular degeneration was detected, and in two of
these animals moderate or severe crystalline deposits were observed.
Also, creatinine and urea plasma levels were substantially increased at
approximately the same levels as for AZ’7847. However, there were
no changes in plasma chemistry parameters in the low-dose group
with AZ’0908. Crystals in the urine were found in both dose groups.
Fig. 6. Metabolic profiling of AZ’4270 and AZ’4284 in rat hepatocytes. (A) LC-MS chromatogram of a rat hepatocyte incubation of AZ’4270. The chromatogram is
showing the combined base peak intensities of the molecular ion [M-H]2 of the parent molecule m/z (mass-to-charge ratio) 517.071 and the potential metabolites with m/z
values of 297.073, 299.089, 315.084, 395.041, 475.121, 515.055, 531.050, 533.066, 549.061, 571.073, 613.022, 709.098, 838.135, and 840.129. The metabolic profiling
was confirmed by radio monitoring detection of [14C]-AZ’4270 (not shown). (B) LC-MS chromatogram of a rat hepatocyte incubation of AZ’4284. The chromatogram is
showing the combined base peak intensities of the molecular ion [M-H]2 of the parent molecule m/z 433.034 and the potential metabolites with m/z values of 213.036,
215.052, 231.047, 311.003, 391.0084, 419.018, 431.018, 433.034, 435.013, 435.049, 447.013, 449.028, 465.028, 467.039, 487.035, 528.985, and 625.061. Putative peak
identification was suggested by LC-MS analysis with accurate mass determination. P = parent molecule, and differences in m/z value in relation to parent m/z value are shown
for major peaks.
Downloaded from dmd.aspetjournals.org at ASPET Journals on July 31, 2017
Fig. 5. Amide hydrolysis of AZ’7847. Amide hydrolysis results in the formation of
two metabolites: M1 and Hyd. The position of the [14C]-labeling is depicted in the
structure.
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Bylund et al.
TABLE 3
Toxicokinetic summary of toxicity study 2
Day 1
Compound
Dose
No. of
Animals
mmol/kg
AZ’7847
M1
M1
AZ’4270
AZ’4270
AZ’4284
AZ’4284
1000
500
2000
170
500
70
200
2
2
2
2
2
2
2
Cmax
Parent
AUC0–24
Parent
Cmax
M1
AUC0–24
M1
mM
mMh
mM
mMh
105
NA
NA
170
300
50
90
1370
NA
NA
1260
3410
610
970
95
57
66
8
19
1.6
3.1
1730
1075
1150
125
330
23
54
Day 7
AUC0–24 M1/AUC0–24
Parent (ratio)
1.3
NA
NA
0.10
0.10
0.04
0.06
Cmax
Parent
AUC0–24
Parent
Cmax
M1
AUC0–24
M1
mM
mMh
mM
mMh
95
NA
NA
190
320
50
110
990
NA
NA
1600
3010
550
1070
310
70
80
10
30
2
5
5220
1300
1250
150
500
30
75
AUC0–24 M1/AUC0–24
Parent (ratio)
5.3
NA
NA
0.09
0.17
0.05
0.07
AUC0-24
Parent/Dose
0.99
2.6
0.63
9.4
6.0
7.9
5.4
AUC0–24, area under the curve for 24 hours; NA, not available.
Discussion
Fig. 7. Rat PK analysis of AZ’0908 and M1 formation. PK curve for rats
administrated with a single dose of 10 mmol/kg. Traces marked with triangles
represent AZ’0908 and traces marked with circles represent the M1 metabolite.
Exposure data are shown in Table 4.
TABLE 4
Pharmacokinetic and toxicokinetic summary of AZ’0908
Day 1
Dose
10 mmol/kg
435 mmol/kg
900 mmol/kg
No. of
Animals
3
5
5
Anticipated
AUCther
Cmax
Parent
AUC0–24
Parent
Cmax
M1
AUC0–24
M1
mMha
mM
mMh
mM
mMh
118
118
118
11
375
540
58
2220
5030
0.6
NA
NA
11
NA
NA
Day 14
AUC0–24 M1/AUC0–24
Parent (ratio)
AUC0–24
Parent/Dose
Cmax
Parent
AUC0–24
Parent
mM
mMh
0.2
NA
NA
5.8
5.1
5.6
NA
415
480
NA
2520
5000
AUC0–24 Parent/
AUCther (ratio)
AUC0–24
Parent/Dose
NA
21
42
NA
5.8
5.6
AUC0–24, area under the curve for 24 hours; AUCther, area under plasma concentration time curve needed for therapeutic effect; NA, not available.
a
AUCther was based on the assumption that a steady-state concentration (Css) average corresponding to 10 times the AZ’0908 whole blood assay IC50 values was needed.
Downloaded from dmd.aspetjournals.org at ASPET Journals on July 31, 2017
This study is an example of problem solving in a drug project
encumbered with kidney toxicity in rats. The mPGES-1 inhibitor
AZ’7847 intended for pain treatment was assessed in a 7-day toxicity
study in rats and was found to induce kidney injury at exposures
representing less than 4 times the anticipated efficacious exposure in
humans. Because AZ’7847 exhibited no activity on the rat mPGES-1
target (not shown), it was unlikely that the observed toxicity was
related to target activity. Instead, the characteristic kidney damage and
the identification of crystal deposits suggested that the kidney toxicity more likely was caused by tubular precipitation of AZ’7847associated drug material. Based on metabolic profiling experiments,
we hypothesized that the crystal deposits were composed of a bis-
sulfonamide metabolite (M1) associated with amide hydrolysis. To
test the hypothesis, rats were given the M1 metabolite and two low
potency mPGES-1 inhibitor analogs, where amide hydrolysis was
undetectable in rat hepatocyte experiments. The results suggested that
compounds with a reduced propensity to undergo amide hydrolysis
and thus having less ability to form M1 reduced the risk of inducing
kidney toxicity. Additional support for our hypothesis of M1 induced
kidney toxicity can be found in a recent methodology study (Nilsson
et al., 2012) where it was confirmed that the crystal deposits in rat
kidneys formed after AZ’7847 administration were composed of the
M1 metabolite. This was realized by using nuclear magnetic resonance
analysis and matrix-assisted laser desorption ionization mass spectrometry imaging.
Rats treated with M1 alone surprisingly showed no histopathologic
changes in the kidney. However, TK analysis revealed low exposure
levels and non-dose-proportionate kinetics for the M1 metabolite. This
was likely related to absorption limitations, as the solubility of the M1
metabolites was poor. The lack of kidney toxicity in rats treated with
the M1 metabolite alone was likely due to underexposure. It has been
shown that Cmax is important for the development of kidney injury
associated with drug precipitation for acyclovir, as rapid bolus
injections lead to an increased risk of acute renal failure as compared
with slow infusions of the same dose (Gnann et al., 1983). However,
in this case, when the M1 metabolite is released from the parent
molecule, the M1 Cmax appears to be directly related to its exposure.
The threshold for M1-induced rat kidney injury appeared to be rather
high. Exposure to M1 at approximately 1300 mMh and Cmax of 80 mM
generated visual crystals in the urine, but neither changes in kidney
functional biomarkers nor detectable histopathologic changes occurred. A comparison of M1 exposure between day 1 and day 7 clearly
showed an accumulation over time in rats treated with AZ’7847. In
rats treated with M1 alone, AZ’4270 or AZ’4284, accumulation was
significantly less or not obvious. This is likely related to severely
Amide Hydrolysis-Induced Kidney Toxicity
Acknowledgments
The authors thank Thomas Meijer, Karin Tunblad, Daniel Jansson, Sveinn
Briem, and Johan Sandell for their contributions, and Magnus Halldin for
valuable scientific consultation.
Authorship Contributions
Participated in research design: Bylund, Annas, Hellgren, Andersson,
Svanhagen.
Conducted experiments: Bylund, Annas, Hellgren, Bjurström, Andersson,
Svanhagen.
Performed data analysis: Bylund, Annas, Hellgren, Bjurström, Andersson,
Svanhagen.
Wrote or contributed to the writing of the manuscript: Bylund, Annas,
Andersson, Svanhagen.
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Address correspondence to: Johan Bylund, Drug Metabolism and Pharmacokinetics, CNSP iMed Science, AstraZeneca R&D, Innovative Medicines, SE-15185
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decreased kidney function in the rats that received 1000 mmol/kg
AZ’7847.
Despite the lack of detection of amide hydrolysis by their corresponding carboxylic acids in vitro, the tool compounds AZ’4270 and
AZ’4284 underwent, to some extent, amide hydrolysis in vivo as
judged by the detection of the corresponding M1 metabolite in plasma.
Amide hydrolysis can be catalyzed by several different hydrolases
in different tissues (Testa and Krämer, 2007). It is possible that
amide hydrolysis in vivo of AZ’4270 and AZ’4284 to a large extent
was carried out by extrahepatically expressed enzymes in the rat.
Nevertheless, there appeared to be a correlation between the degree
of hydrolysis in vivo and the propensity to undergo amide hydrolysis in rat hepatocytes in vitro. Interestingly, amide hydrolysis might
be for the most part a rat-specific phenomenon. In human and dog
hepatocytes, amide hydrolysis could not be detected for AZ’7847
as its corresponding carboxylated acid metabolite (Hyd). In addition, human hepatocyte experiments suggested that the compound
AZ’7847 was more stable in vitro when compared with rats. It is
therefore possible that M1-induced kidney toxicity after treatment
of mPGES-1 inhibitors of this chemical series would not pose a
major problem in humans because amide hydrolysis occurrence is
likely lower and the doses needed to induce toxicity would be
unattainable for all practical purposes. However, identification of
a rodent toxicity species would be essential for progression into
clinical trials; therefore, an extensive effort was conducted to identify
an mPGES-1 inhibitor candidate that could achieve larger margins in
the rat.
A novel and potent mPGES-1 inhibitor, AZ’0908, with a low
affinity for amide hydrolysis and thus improved rat PK was identified.
This was likely achieved by structural modifications, which resulted in
reduced amide hydrolysis. In a 14-day rat toxicity study, AZ’0908
exhibited a margin that was 21-fold the anticipated AUCther in human.
As a result of plasma deficiency due to microsampling, the M1
metabolite was not monitored in this study. However, for AZ’0908 or
any other compound of this chemical series to progress into clinical
trials, margins based on exposures of both the parent molecule and the
M1 metabolite would have to be established in future toxicity studies.
Although in vitro results indicate that the M1 formation is less
pronounced in humans than in rats, uncertainties exist concerning the
intraspecies translational aspects of M1 formation. Therefore, future
clinical trials that include compounds of this chemical series would
need to exercise caution regarding the initial administration of parent
compound and its exposure in relation to M1.
In conclusion, we have identified a novel mPGES-1 inhibitor that
can be toxicologically evaluated in the rat. This was achieved by
exploratory studies that linked together aspects of both drug metabolism
and toxicity.
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