0090-9556/99/2711-1367–1373$02.00/0
DRUG METABOLISM AND DISPOSITION
Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics
Vol. 27, No. 11
Printed in U.S.A.
SUBSTANCE P RECEPTOR ANTAGONIST I: CONVERSION OF PHOSPHORAMIDATE
PRODRUG AFTER I.V. ADMINISTRATION TO RATS AND DOGS
SU-ER W. HUSKEY, DEBRA LUFFER-ATLAS, BRIAN J. DEAN, ERIN M. MCGOWAN, WILLIAM P. FEENEY,
SHUET-HING LEE CHIU
AND
Department of Drug Metabolism (S.-E.W.H., D.L.-A., B.J.D., E.M.M., S.-H.L.C.) and Laboratory Animal Resources (W.P.F.), Merck Research
Laboratories. Rahway, New Jersey
(Received February 26, 1999; accepted August 10, 1999)
This paper is available online at http://www.dmd.org
ABSTRACT:
was rapid after i.v. dosing of I to rats and dogs. The relative extent
of exposure of II after i.v. dosing of I was estimated by comparing
the dose-adjusted area under the plasma concentration versus
time curve values of II after i.v. dosing of I with those after i.v.
dosing of II. In rats, the extent of exposure was estimated to be
;90 and ;100% at 1 and 8 mg/kg, respectively; in dogs, that was
;59% at 0.5 mg/kg. A nonproportional increase in the area under
the concentration versus time curve value of II with dose was
observed after i.v. administration of I in dogs from 0.5 to 32 mg/kg,
suggesting that the elimination of II might have been saturated at
higher doses.
In recent years, the successful cloning and expression of human
tachykinin receptors have initiated an intensive search for selective
nonpeptide receptor antagonists in the pharmaceutical industry using
in vitro receptor binding assays (Takeda et al., 1991; Cascieri et al.,
1992; Fong et al., 1992). The biological actions of the tachykinins are
mediated through specific cell-surface receptors. Three subtypes, designated as NK1, NK2, and NK3, have been identified on the basis of
marked differences in the rank order of potencies of agonist peptides
in different tissues, with Substance P being the preferred agonist for
NK1 receptors, neurokinin A for NK2 receptors, and neurokinin B for
NK3 receptors (Maggi et al., 1993; Otsuka and Yoshioka, 1993).
Evidence to date suggests that Substance P acting via the NK1
receptor may be involved in the pathoetiology of emesis (Andrews et
al., 1988; Bountra et al., 1993; Tattersall et al., 1994, 1996; Hale et al.,
1996; Kris et al., 1997), migraine (Perianin et al., 1989; Moussaoui et
al., 1993; Longmore et al., 1995), pain (Otsuka and Yanigasawa,
1988), and depression (Kramer et al., 1998).
Several pharmacophores have been used in the synthesis of NK1
receptor antagonists, including quinuclidine (Snider et al., 1991; OuryDonat et al., 1993), piperidine (McLean et al., 1993, 1996; Stevenson et
al., 1995; Ward et al., 1995; Armour et al., 1996; Gardner et al., 1996;
Gonsalves et al., 1996; Ladduwahetty et al., 1996; Rosen et al., 1998),
tryptophan (MacLeod et al., 1994), diacylpiperazine (Mills et al., 1993),
pyrido[3,4-b]pyridine (Natsugari et al., 1995), and morpholine (Hale et
al., 1996, 1998). L-754,030 (II)1 (Fig. 1) is a very potent reversible NK1
receptor antagonist (M.A. Cascieri, unpublished data) (Kd 5 86 pM) of
the morpholine series (Cascieri et al., 1997); however, II exhibits limited
solubility in aqueous buffers (J.V. Pivnichney and D.A. Levorse, unpublished data; H. Jahansouz and M.L. Bray, unpublished data) (;8 mg/ml
at pH 8), which presents a challenge for formulation as an i.v. drug for
antiemesis (Rupniak et al., 1997). A prodrug approach was thus taken in
chemical synthesis to increase the solubility of II (Benkovic and Sampson, 1971; Anderson et al., 1985), and among prodrug derivatives synthesized, L-758,298 (I), a phosphoramidate prodrug of II, exhibits the
best overall profile of a prodrug including solubility and in vivo rate of
conversion. Compound I is a relatively weak antagonist for human NK1
receptor (M.A. Cascieri, unpublished data) (Kd 5 4 nM); it is freely
soluble in aqueous buffers at a solubility of ;55 mg/ml (free acid
equivalents at pH 8) (J.V. Pivnichney and D.A. Levorse, unpublished
data; H. Jahansouz and M.L. Bray, unpublished data), which is about a
7000-fold increase in aqueous solubility compared with II. Moreover,
compound I can be hydrolyzed to II chemically under mild acidic
Send reprint requests to: Dr. Su-Er W. Huskey, Dept. of Drug Metabolism,
Merck Research Laboratories, P.O. Box 2000, Rahway, NJ 07065. E-mail:
[email protected]
1
Abbreviations used are: L-754,030 (II), [(2R)-((1R)-3,5-bis(trifluoromethylphenyl)ethoxy)-(3S)-(4-fluoro)phenyl-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methylmorpholine]; L-758,298 (I), [(2R)-(1-(R)-3,5-bis(trifluoromethyl)phenylethoxy)-3-(S)-(4fluoro)phenyl-4-(3-(1-phosphoryl-5-oxo-4H-1,2,4-triazolo)methylmorpholine;
Compound III, [(2R)-(3,5-bis(trifluoromethyl)benzyloxy)-(3S)-phenyl-4-(3-(1phosphoryl-5-oxo-4H-1,2,4-triazolo)methylmorpholine; Compound IV, [(2R)-((1R)3,5-bis(trifluoromethylphenyl)ethoxy)-(3S)-phenyl-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methylmorpholine]; AUC, area under the plasma concentration versus time
curve; PEG400, polyethylene glycol; SD, Sprague-Dawley; LC-MS/MS, liquid chromatography-tandem mass spectrometry; SRM, selected reaction monitoring; Vdss,
volume of distribution at steady state.
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A water-soluble phosphoramidate prodrug (L-758,298, compound
I) of the potent and selective human Substance P receptor antagonist L-754,030 (compound II) is under development as an i.v. drug
for treatment of emesis, migraine, and chronic pain. Compound I
undergoes hydrolysis readily to II under acidic conditions. In the
studies reported herein, we investigated the stability of I in blood
and hepatic subcellular fractions from rats, dogs, and humans as
well as the conversion of I to II in rats and dogs after i.v. dosing.
Compound I was converted to II rapidly in rat blood but was stable
in dog and human blood. However, the conversion was rapid in
liver microsomes prepared from dogs and humans. As expected
from the results of in vitro studies, the in vivo conversion of I to II
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HUSKEY ET AL.
conditions (J.V. Pivnichney and D.A. Levorse, unpublished data; H.
Jahansouz and M.L. Bray, unpublished data) or enzymatically via the
action of alkaline phosphatase (M.A. Cascieri, unpublished data; S.E.W.H. and B.J.D., unpublished data). This report describes the in vitro
stability of I in blood and its metabolic stability in subcellular fractions of
preclinical species (rat and dog) and human. In addition, studies conducted to investigate the pharmacokinetics of I and its conversion after
i.v. dosing in rats and dogs are presented in this report.
Materials and Methods
Chemicals. L-758,298 (I; bis-N-methyl-D-glucamine salt or dipotassium
salt), L-754,030 (II; free base), compound III (bis-N-methyl-D-glucamine salt
or dipotassium salt), and compound IV (free base) were synthesized and the
synthetic procedures were published separately (Fig. 1) (Hale et al., 1998).
Compounds III and IV were used as the internal standard for the quantification
of I and II, respectively. Sodium vanadate, polyethylene glycol (PEG400), and
propylene glycol were purchased from Fisher Scientific (Springfield, NJ). All
other chemicals were reagent or HPLC grade and were purchased from EM
Science (Gibbstown, NJ).
Validation of a Procedure for Blood and Plasma Sample Preparation.
Fresh heparinized blood from male Sprague-Dawley (SD) rats or male beagle
dogs was divided into 1- or 1.4-ml aliquots to which prodrug I was added in
duplicate at concentrations ranging from 25 ng/ml to 25 mg/ml or 35 ng/ml to
70 mg/ml, respectively. Sodium vanadate (final 5 mM, 25 or 35 ml of 200 mM)
was added immediately to one set of blood samples whereas saline (35 ml) was
added to the other set of blood samples. The blood samples were kept on ice
for 30 min, then centrifuged at 4°C for 15 min. The resulting plasma (0.2 ml)
from each tube was transferred to clean tubes, IV (60 ng) was added as the
internal standard for II, and the samples were processed immediately by
solid-phase extraction and analyzed by liquid chromatography-tandem mass
spectrometry (LC-MS/MS) for II.
Stability of I in Blood from Rat, Dog, and Human. Fresh heparinized
blood from male SD rats, male beagle dogs, or humans (from two male
subjects) was divided into 0.5-ml aliquots to which compound I was added in
triplicate at a concentration of 1 or 10 mg/ml. Blood samples were incubated
at 37°C for 15, 30, 60, and 120 min. At these specified time intervals, 12.5 ml
of 200 mM vanadate solution was added immediately to each incubate and the
sample was kept on ice for 5 min and centrifuged at 4°C for 10 min. The
resulting plasma from each tube (0.2 ml) was transferred to a clean tube
containing 50 ng of III and 50 ng of IV, the respective internal standards for
I and II; this was followed by the addition of 1.7 ml of water and 0.5 ml of
acetonitrile. The mixture was processed immediately by solid-phase extraction
and analyzed by LC-MS/MS for the concentrations of I and II simultaneously.
The storage time for samples was less than 1 week.
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FIG. 1. Structures of compounds I, II and two internal standards, III and IV.
Metabolic Stability of I in Subcellular Fractions of Dog and Human
Liver. All liver microsomes and cytosolic fractions were prepared using the
following procedure. Thawed livers were homogenized with 2 volumes of 50
mM Tris-buffer (pH 7.5) containing 1.15% KCl. For microsomal and cytosolic
preparations, the homogenate was centrifuged for 20 min at 9000g and the
resulting supernatant was centrifuged for 60 min at 105,000g. The resulting
cytosolic fractions were recentrifuged at 105,000g for 60 min. Fractions of
cytosol were aliquoted into small tubes and stored at 270°C. Subsequently, the
microsomal pellets were washed with 10 mM EDTA containing 1.15% KCl
and were centrifuged at 105,000g for 60 min. Washed microsomes were
resuspended in 10 mM potassium phosphate buffer (pH 7.4) containing 250
mM sucrose, aliquoted into small tubes, and stored at 270°C. Protein concentrations were determined by a modified Lowry assay (Smith et al., 1985).
The specific cytochrome P-450 content in each microsomal preparation was
measured as described by Omura and Sato (1964).
Frozen cytosolic and microsomal fractions (stored at 270°C) of dog (preparation 116) and human livers (Nos. 113, 115, and 118) were used in the in
vitro studies. Human liver was obtained from Professor W.G. Levine (Department of Molecular Pharmacology, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY). The organ donors were a 46-year-old male
(113), a 59-year-old male (115), and a 43-year-old male (118), all with no
known drug history. The microsomal or cytosolic fractions from human liver
were combined with equal amounts of proteins from three subjects (Nos. 113,
115, and 118).
Compound I (final concentration 8.1 mM) was incubated in triplicate with
cytosolic or microsomal fractions (0.5 mg/ml) of dog or human liver at 37°C for
15, 30, 60, and 120 min. The reaction was quenched by the addition of acetonitrile
(0.5 ml) and two internal standards, kept on ice, followed by the addition of 1.7 ml
of water (final concentration of solvent ;18%). The mixture was processed
immediately by solid-phase extraction and analyzed by LC-MS/MS for I and II
simultaneously. The storage time for samples was less than 1 week.
Dose Preparation. The doses for compound I were prepared by dissolving
I (the bis-N-methyl-D-glucamine salt, molecular weight 1004.9) in a solution of
lactose (50 mg/ml), potassium carbonate (1.38 mg/ml), citric acid monohydrate
(0.85 mg/ml), and sodium chloride (4 mg/ml) (pH 7.0). Doses were filtered
through a 0.45 mm filter before dosing. The doses for II were prepared in a
solution of ethanol/propylene glycol/water (15:60:25, v/v/v) or in a solution of
ethanol/PEG400/water (20:60:20, v/v/v). Doses of II were prepared and stirred
constantly at 25°C overnight before dosing.
Pharmacokinetics in Rats. Male SD rats were obtained from Charles River
Breeding Laboratories (Wilmington, MA) or Taconic Laboratories (Germantown, NY). They were housed under standard conditions and were maintained
under a 12-h light/dark cycle in the Laboratory Animal Resources facilities,
Merck Research Laboratories, Rahway, NJ. They were allowed access to
commercial rodent chow and water ad libitum. Rats were fasted overnight
before dosing and then until 1 h after dosing. Water was allowed ad libitum
during the fasting period.
Rats were cannulated at the femoral vein for serial bleeding and jugular vein
for dosing. The b.wt. of individual rats, ranging from 0.3 to 0.4 kg, were
determined on the morning of the study. Four male rats per group were dosed
i.v. with I or II by bolus injection into the jugular vein at 1, 8, or 25 mg/kg body
weight or at 0.2, 2, or 5 mg/kg b.wt., respectively. After dosing, 0.5-ml
specimens of blood were collected by serial sampling from the femoral cannula
at 2 to 3, 5, 15, and 30 min, and 1-ml specimens of blood were drawn at 1, 2,
4, 6, 8, 10, 24, 30, 48, and 72 h. After 1 h, blood was replaced with an equal
volume of sterile heparinized saline and donor blood.
Pharmacokinetics in Dogs. Six male Beagle dogs were housed under
standard conditions and were maintained under a 12-h light/dark cycle in the
Laboratory Animal Resources facilities, Merck Research Laboratories, Rahway, NJ. They were allowed access to water ad libitum. Dogs were fasted
overnight before dosing and then until 4 h after dosing. Water was allowed
during the fasting period. The b.wt. of the individual dogs ranged from 9.6 to
14.3 kg.
The dogs were dosed i.v. with I or II by bolus injection into the cephalic vein
via an indwelling vascular catheter at 0.5 or 2 mg/kg b.wt. or at 0.2, 0.5, and 2
mg/kg body weight, respectively. At the 32 mg/kg dose of I, the dogs were dosed
by infusion into the cephalic vein via an indwelling vascular catheter for 45 s at 3.2
ml/kg followed by a saline flush for 15 s. After dosing, heparinized blood samples
CONVERSION OF A PHOSPHORAMIDATE PRODRUG IN RATS AND DOGS
tifiable level were treated as zero for the purpose of calculating mean concentrations.
Results
Validation of a Procedure for Blood Sample Preparation. To
determine whether vanadate can prevent the ex vivo conversion of
prodrug (compound I) to II in blood samples, compound I (25 ng/
ml–25 mg/ml) was added to rat or dog blood in the presence of saline
or vanadate (5 mM, an inhibitor for alkaline phosphatase) and the
formation of II was quantified by LC-MS/MS. No significant increase
of concentrations of II was detected in rat blood while stored on ice up
to 1 h (data not shown). When rat blood samples were treated with
saline, 18 to 1800 ng/ml of II (or 10 –13% of I added) was detected in
plasma. In comparison, only 16 to 170 ng/ml of II (or 1–3% of
conversion) was detected when vanadate was added immediately to
rat blood samples after mixing with I at various concentrations to rat
blood (data not shown). In dog blood, the ex vivo conversion of I to
II was low (1–3%), and the addition of vanadate did not achieve
significant reduction of the conversion.
In Vitro Conversion of Prodrug I to II. When the prodrug,
compound I, was added to blood at concentrations of 1 and 10 mg/ml,
incubations were carried out immediately and processed as described
in Materials and Methods. Vanadate was included in the sample
preparation procedure after incubation to minimize the ex vivo conversion of I to II. The concentrations of both compounds in plasma at
selected time intervals were determined simultaneously by LC-MS/
MS. The conversion of I to II in rat, dog, or human blood was
expressed by plotting the increase in molar concentration of II against
the decrease in molar concentration of I in plasma versus time (Fig. 2).
The concentrations of I in rat plasma diminished rapidly with a
half-life of ;30 min when 10 mg/ml of I was added (Fig. 2A). Similar
results were obtained from incubations with I at 1 mg/ml (data not
shown). Compound I was more stable in dog blood than in rat blood
with a half-life (in dog blood) of ;230 and ;350 min for 1 and 10
mg/ml, respectively; only 20 to 30% of I was converted to II during
the 2-h incubation period (Fig. 2B). Compound I was very stable in
human blood with less than ;15% conversion observed during the
2-h incubation period (Fig. 2C).
Metabolic Stability of Compound I in Subcellular Fractions of
Dog and Human Liver. Compound I (5 mg/ml) was incubated with
microsomal and cytosolic fractions followed by simultaneous quantification of compound I and its dephosphorylated product (compound
II) by LC-MS/MS. The decline of its concentrations with the concomitant increase of the levels of compound II with time is shown in
Figs. 3 and 4. In dog liver microsomes, the conversion was nearly
complete in 30 min with only ;5% of the substrate remaining at that
time. The conversion of I to II was rapid in human liver microsomes
such that only ;2% of I was detectable after the first time point (15
min); the conversion was complete at 30 min. The rate of conversion
was slower in cytosolic fractions for both species (55% remaining at
30 min in dog; 78% remaining in human). After the 2-h incubation
period, conversion by hepatic cytosolic fractions was nearly complete
(; 6% remaining) in dog, and ;65% complete (;35% remaining) in
human.
In Vivo Conversion of I in Rats. The conversion of I to II was
studied in rats dosed i.v. at 1, 8, and 25 mg/kg b.wt. The concentrations of I and II in plasma samples were determined simultaneously by
LC-MS/MS (method 2). The concentrations of intact I were quantifiable only at the earliest time points (2–5 min) for the lowest dose,
and up to 1 h for the highest dose. After the 25 mg/kg dose, the
concentrations of I in plasma declined rapidly from 1117 to 75 ng/ml
between 3 and 60 min (data not shown).
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(5 ml) were collected by serial bleeding from the jugular vein at 2.5 (or 3), 5 (or
6), 15, 30 min, 1, 2, 4, 6, 8, 10, 24, 30 (only at 0.5 mg/kg), 48, and 72 h.
Sample Preparation. To minimize enzymatic hydrolysis of I in blood
during sample preparation, 12.5 to 125 ml of 200 mM vanadate in saline (final
concentration 5 mM) was added immediately to fresh blood samples, which
were kept on ice. Plasma was obtained by centrifugation at 4°C within 30 min.
Each plasma sample (0.2 ml for rats and 0.5 ml for dogs) was added to a test
tube containing 60 to 250 ng of III and 60 to 250 ng of IV, the respective
internal standards for I and II, followed by 1.7 ml of water and 0.5 ml of
acetonitrile. The mixture then was loaded onto a Varian BondElut C18 cartridge (500 mg). The cartridge was washed with ;6 ml of water followed by
elution with ;3 ml of methanol. The methanol eluent was evaporated to
dryness and stored at 4°C (storage time less than 1 week) before analysis using
a LC-MS/MS assay.
Quantification of I and II in Plasma by LC-MS/MS. The quantification
of I and II in plasma was performed on a SCIEX API III tandem mass
spectrometer using the ionspray interface. The collision gas used for collisioninduced dissociation was argon. The HPLC system consisted of two Shimadzu
10AD pumps, SCL-10A controller and SIL-10A autoinjector. Chromatographic separation was performed on a BDS-Hypersil C18 column (4.6 3 250
mm) using a mobile phase consisting of 72% acetonitrile and 28% 10 mM
ammonium acetate (adjusted to pH 7.4 with HPLC grade triethylamine). The
flow rate was 1.05 ml/min and the effluent was split such that 5% of the flow
entered the ionspray interface. In this system, I and its internal standard, III,
eluted at approximately 2.2 min; II and its internal standard, IV, eluted at
approximately 4.4 and 4.6 min, respectively.
A two-period acquisition method was used due to the significant difference
in retention time and peak shape of the two sets of compounds. The first period
used a dwell time for selected reaction monitoring (SRM) of 475 ms with a
10-ms pause; the dwell time for the second period was 400 ms with a 5-ms
pause. Positive ion detection was used during data acquisition for II and its
internal standard; however, both positive and negative ion detection were used
in different cases for the detection of I and its internal standard.
A two-period SRM assay (method 1) was developed, with negative ion
detection for I and III in the first period, followed by positive ion detection for
II and IV in the second period. In the first period, the negative precursor/
product ion pairs at m/z 5 613/79 and 581/79 were used for quantification of
I and III, respectively; in the second period, the positive precursor/product ion
pairs at m/z 5 535/277 and 517/259 were used for quantification of II and V,
respectively.
Alternatively, a two-period positive ion SRM assay (method 2) was used
subsequently to eliminate cross talk between the channels. In the first period,
the precursor/product ion pairs at m/z 5 615/277 and 583/259 were used for
quantification of I and III, respectively; in the second period, the precursor/
product ion pairs at m/z 535/179 and 517/161 were used for quantification of
II and IV, respectively.
Two standard curves were generated for each assay by plotting the peak area
ratio of the response for either I or II to that of its respective internal standard
versus the amount of compound added to the control plasma sample. The range
of concentrations used to define the standard curve was dependent on the
expected plasma levels, and the amount of internal standard used was chosen
to be roughly at the mid-point of the standard curve. An average of three
replicates at each concentration over the entire range was used in rat and dog
plasma to establish the standard curves. A power fit regression [ Y 5 kXn ] was
used to quantify the unknowns. The limits of quantification for I were 6.25 to
62.5 ng/ml of rat plasma and 25 to 100 ng/ml of dog plasma; limits of
quantification for II were 6.25 to 12.5 ng/ml of rat plasma and 5 to 20 ng/ml
of dog plasma.
Calculations of Pharmacokinetic Parameters. The area under the plasma
concentration versus time curve (AUC) was determined by the UNICUE
program with linear trapezoidal interpolation in the ascending slope and
logarithmic trapezoidal interpolation in the descending slope (Yeh and Small,
1989). The portion of the AUC from the last measurable concentration of II in
plasma to infinity was estimated by Ct/l, where Ct represents the last measurable concentration in plasma and l is the terminal rate constant determined
from the plasma concentration versus time curve by linear regression at the
elimination phase of the semilogarithm plot. Concentrations below the quan-
1369
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HUSKEY ET AL.
FIG. 2. Stability of prodrug I in rat, dog, and human blood at 10 mg/ml.
I (10 mg/ml; MW 614.4 as free base) was incubated with fresh heparinized rat
(A), dog (B), human (C) blood at 37°C for 0 to 120 min. After incubation, vanadate
(5 mM) was added immediately and the samples were kept on ice to minimize any
ex vivo hydrolysis of I. Plasma was obtained by centrifugation at 4°C, and 0.2 ml
of the resulting plasma was mixed with internal standard, processed by solid-phase
extraction, and analyzed simultaneously for I and II by LC-MS/MS.
The concentration of II in plasma was maximal at the first sampling
time point (2–3 min) after i.v. dosing of I at all three dose levels (Fig.
5). Ten hours after dosing with I at 1, 8, and 25 mg/kg, the plasma
concentrations of II were ;14, ;140, and ;620 ng/ml, respectively;
at 24 h, the levels were measurable only in one rat given the highest
dose (25 mg/kg).
A near proportional increase in the AUC values of II with dose was
observed after i.v. administration of I at 1 and 8 mg/kg (Table 1). At 25
mg/kg, the AUC value increased ;4-fold over that at 8 mg/kg. The
elimination curve (Fig. 5) showed a convex phase (2–10 h) at the highest
concentration, and the factors contributing to this phenomenon is uncertain.
Pharmacokinetics of II also were studied in rats dosed i.v. with II at
0.2, 2, and 5 mg/kg. As shown in Fig. 6, a steady decline of II was
observed after all three doses. The kinetics appeared to be linear over
the dosing range, with an increase in plasma AUC values nearly
proportional to the 10- and 25-fold increase in dose from 0.2 to 2 and
5 mg/kg, respectively. Plasma clearance was ;15 ml/min/kg, the
volume of distribution at steady state (Vdss) was ;3 liters/kg, and the
terminal half-life was ;3 h (Table 1).
The AUC values of II in rat plasma after dosing of I or II were
compared to estimate the relative extent of exposure of II. Due to the
large differences in the molecular weights (I, mw 1004.9 salt; II, mw
534.4 free base), the AUC values were normalized to per mole of
dose. When rats were dosed with I at 1 mg/kg, the relative extent of
exposure of II in plasma was estimated to be ;91% by comparison
with the average AUC calculated from the 0.2 and 2 mg/kg i.v. doses
of II. Similarly, when the dose was 8 mg/kg, relative extent of
exposure of II in plasma was ;100%, estimated by comparing the
average AUC values calculated from the 2 and 5 mg/kg i.v. doses of
II. At the highest dose (25 mg/kg), the relative extent of exposure was
not determined because plasma concentrations and AUC values of II
exceeded those from the highest i.v. dose (5 mg/kg) of II and therefore
could not be compared with one another.
In Vivo Conversion of I in Dogs. The in vivo conversion and
pharmacokinetics of I were studied in beagle dogs dosed i.v. at 0.5, 2,
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FIG. 3. Metabolic stability of I in subcellular fractions of dog liver.
I (8.1 mM) was incubated with microsomal or cytosolic fractions of dog liver at
37°C for 0 to 120 min. After incubation, samples were processed by solid-phase
extraction and analyzed simultaneously for I and II by LC-MS/MS.
1371
CONVERSION OF A PHOSPHORAMIDATE PRODRUG IN RATS AND DOGS
TABLE 1
Conversion of I to II in plasma of rats dosed i.v. with Compound I or II
i.v. Dose of Compound I (mw 1004.9 salt)
Dose
1
8
25
Dose (mmol/kg)
AUC of II (ngzh/ml)
1.00
568 6 121
7.96
6249 6 1373
24.88
22392 6 1158
Exposure of II (%)a
;91%
;100%
NCb
mg/kg
i.v. Dose of Compound II (mw 534.4 free base)
Dose
0.2
2
5
mg/kg
Dose (mmol/kg)
AUC of II (ngzh/ml)
Clearance (ml/min/kg)
Vdss (liters/kg)
T1/2c (h)
0.37
201 6 52
17.6 6 5.3
3.2 6 0.5
2.7 6 0.4
3.74
2658 6 335
12.7 6 1.7
2.8 6 0.1
3.2 6 0.4
9.36
6377 6 1242
13.4 6 2.6
2.5 6 0.2
2.4 6 0.4
FIG. 4. Metabolic stability of I in subcellular fractions of human liver.
I (8.1 mM) was incubated with microsomal or cytosolic fractions of human liver
at 37°C for 0 to 120 min. After incubation, samples were processed and analyzed by
LC-MS/MS.
FIG. 6. Mean (6 S.D.) concentrations of II in plasma of male rats dosed i.v.
with II.
Male SD rats (n 5 4) were i.v. dosed with II (mw 534.4 free base) prepared in
a solution of ethanol/propylene glycol/water (15:60:25, v/v/v) or in a solution of
PEG400/water/ethanol (60:20:20, v/v/v). Plasma samples (0.2 ml) were processed
by solid-phase extraction and analyzed for II by LC-MS/MS. The limit of quantification for II was 0.5 to 1.25 ng/ml.
FIG. 5. Mean (6 S.D.) concentrations of II in plasma of male rats dosed i.v.
with I.
Four male SD rats were dosed i.v. with I (bis-N-methyl D-glucamine salt; mw
1004.9 salt) prepared in a solution of lactose, potassium carbonate, citric acid
monohydrate, and sodium chloride (pH 7.0), as described in Materials and Methods.
Plasma samples (0.2 ml) were processed by solid-phase extraction and analyzed
simultaneously for I and II by LC-MS/MS. The limits of quantification for I and II
were 6.25 to 62.5 and 6.25 to 12.5 ng/ml, respectively.
and 32 mg/kg. The concentrations of I and II in plasma samples were
determined simultaneously by LC-MS/MS (method 1). After i.v.
dosing of prodrug I to dogs, conversion to II was very rapid and intact
I levels were measurable only at 2 to 3 min for the two lower doses.
For the highest dose (32 mg/kg), concentrations of I ranged from 32
to 77 mg/ml (mean 57 mg/ml) and declined rapidly to 1.3 mg/ml by 5
to 6 min. The corresponding concentrations of II were ;20 and 17
mg/ml, indicating a much slower decline compared with compound I.
For all three doses studied, II levels in plasma were the highest at the
first time point and declined slowly thereafter (Fig. 7). At 24-h post
dosing, plasma levels of II were 7 to 155 ng/ml for the two lower
doses; at 72 h, the levels of II (;2.6 mg/ml) remained in the quantifiable range only in dogs treated with the highest dose.
A nonproportional increase in AUC values of II with dose was
observed after i.v. administration of I. For the doses of 2 and 32
mg/kg, the AUC value was estimated only through 48 or 72 h due to
nonlinear kinetics and the uncertainty involved in extrapolation from
the last measurable time point to infinity. The AUC value of II
exhibited an 8-fold increase as the dose increased 4-fold from 0.5 to
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 14, 2017
a
Exposure of II was estimated by comparing the AUC values of II in plasma after dosing of
I with that after dosing of II to rats. At 1 mg/kg, the relative exposure was calculated from the
0.2 and 2 mg/kg i.v. doses of II. At 8 mg/kg, the relative exposure was calculated from the 2 and
5 mg/kg i.v. doses of II. Calculations were normalized for dose.
b
NC: not calculated.
c
For T1/2 calculations, the terminal rate constant was estimated by linear regression of the last
three to four data points: 6 to 24 h for 5 mg/kg, 6 to 10 h for 0.2 mg/kg, and 6 to 24 h for 2 mg/kg.
1372
HUSKEY ET AL.
TABLE 2
Conversion of I to II in plasma of dogs dosed i.v. with Compound I or II
i.v. Dose of Compound I (mw 1004.9 salt)
Dose
0.5
2
32
Dose (mmol/kg)
AUC of II (ngzh/ml)
0.50
1093 6 281
1.99
8926 6 1504
31.84
373363 6 55169
Exposure of II (%)a
;59%
NCb
NC
mg/kg
i.v. Dose of Compound II (mw 534.4 free base)
Dose
0.2
0.5
2
mg/kg
FIG. 7. Mean (6 S.D.) concentrations of II in plasma of male dogs dosed i.v.
with I.
2 mg/kg, and a greater than 40-fold increase between 2 and 32 mg/kg
(Table 2). These results suggest that the elimination of II might have
been saturated at the higher doses.
Pharmacokinetics of II also were studied in dogs dosed i.v. with II at
0.2, 0.5, and 2 mg/kg b.wt. A steady decline in the concentration of II in
plasma was observed after i.v. dosing at 0.2 or 0.5 mg/kg (Fig. 8). The
kinetics appeared to be linear over the 0.2 to 0.5 mg/kg dosing range with
a nearly proportional increase in AUC values when the dose increased
2.5-fold. The half-life was 6 to 7 h, plasma clearance was ;2.5 ml/min/
kg, and the Vdss was ;1 liter/kg for the two lower doses (Table 2). When
plasma concentrations from the 2 mg/kg dose were plotted as a function
of time, a convex phase was detected, indicating that the rate of elimination of II was nonlinear in this species at this dose (Fig. 8). The increase
in the plasma AUC0 –72h value was ;10-fold and the decrease in clearance was ;2-fold when the dose was increased from 0.5 to 2 mg/kg
(Table 2). These results suggest that the elimination of II may have been
saturated at the 2 mg/kg dose. The relative extent of exposure of II in
plasma was ;59% at the 0.5 mg/kg dose, as determined by comparing
the plasma AUC value with the average AUC value obtained from i.v.
doses of II at 0.2 to 0.5 mg/kg, at which linear kinetics is followed. Due
to nonlinear kinetics at high concentrations, the relative extent of exposure of II in plasma at the higher doses (2 and 32 mg/kg) could not be
determined (Table 2).
Discussion
To establish assay methods for I in plasma, a procedure was
validated for the preparation of blood samples from rats and dogs.
Vanadate, an inhibitor of alkaline phosphatase, was used to inhibit the
ex vivo hydrolysis of I to II by alkaline phosphatase (Hagerstrand et
al., 1976; Hatoff and Hardison, 1982). The addition of vanadate was
effective in reducing the ex vivo conversion of I to II from 10 to 13%
to 1 to 3% in rat blood, however, its use did not eliminate the
conversion of 1 to 3% in dog blood. Therefore, under the sample
preparation conditions used in our studies, about 1 to 3% of conversion of I to II is expected to take place in both rat and dog blood.
The conversion of compound I, a phosphoramidate prodrug, to the
potent NK1 receptor antagonist, II, was essential for its in vivo
biological activity as a prodrug. This conversion was studied in rat,
0.37
1266 6 95
2.6 6 0.2
1.0 6 0.3
5.7 6 1.4
0.94
3786 6 1117
2.3 6 0.7
1.1 6 0.1
7.3 6 2.6
3.74
37613 6 8037
0.9 6 0.2
0.9 6 0.1
NDd
a
Exposure of II was estimated by comparing the AUC values of II in plasma after dosing of
I with that after dosing of II to dogs. At 0.5 mg/kg, the relative exposure was calculated from the
0.2 and 0.5 mg/kg i.v. doses of II. Calculations were normalized for dose.
b
NC: not calculated.
c
At the 2 and 32 mg/kg doses, the AUC value was calculated up to 72 h because the terminal
rate constants could not be estimated accurately.
d
ND: not determined. The terminal rate constant and T1/2 were not calculated due to the
curvature shown in the plasma concentration versus time profile.
FIG. 8. Mean (6 S.D.) concentrations of II in plasma of male dogs dosed i.v.
with II.
The same three dogs were dosed i.v. with II (mw 534.4 free base) in two studies
and three different dogs (4549, 2804, and 0939) were dosed i.v. with II in one study.
All of the doses were prepared in ethanol/propylene glycol/water (1:6:3, v/v/v).
Plasma samples were processed by solid-phase extraction and analyzed for II by
LC-MS/MS. The limit of quantification for II was 1.0 to 2.5 ng/ml.
dog, and human blood. The conversion was rapid in rat blood,
somewhat slower in dog blood, and very slow in human blood. The
conversion of I to II was further investigated in subcellular fractions
from dog and human liver. Results indicate that compound I was
hydrolyzed rapidly in human and dog liver microsomes. Based on the
above in vitro results, it is anticipated that the conversion of I to II also
will be rapid in preclinical species (rat and dog) and humans, when
compound I is administered i.v.
As expected, the conversion of I to II in vivo was rapid in rats. A
near proportional increase in the AUC values of compound II with
increase in doses of I was observed after i.v. administration to rats at
1, 8, and 25 mg/kg. Pharmacokinetics of compound II appeared to be
linear in rats when it was dosed at 0.2, 2, and 5 mg/kg. As shown in
Table 1, the relative extent of exposure of II in plasma after i.v. dosing
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 14, 2017
Three male beagle dogs were dosed i.v. with I (bis-N-methyl-D-glucamine salt;
mw 1004.9 as salt) prepared in a solution of lactose, potassium carbonate, citric acid
monohydrate, and sodium chloride (pH 7.0). Plasma samples (0.5 ml) were processed by solid-phase extraction and analyzed simultaneously for I and II by
LC-MS/MS with III and IV as the respective internal standards. The limits of
quantification for I and II were 25 to 100 and 5 to 20 ng/ml, respectively.
Dose (mmol/kg)
AUC of IIc (ngzh/ml)
Clearance (ml/min/kg)
Vdss (liters/kg)
T1/2 (h)
CONVERSION OF A PHOSPHORAMIDATE PRODRUG IN RATS AND DOGS
Acknowledgments. We thank Professor W.G. Levine (Department
of Molecular Pharmacology, Albert Einstein College of Medicine,
Yeshiva University, Bronx, NY) for supplying human liver samples;
Dr. R. Stearns, Dr. S. Vincent, Dr. J. Zagrobelny, and C. Lin for
helpful discussions; Dr. J. Jahansouz and M. Bray for supplying
formulation solution; M. Bray and J. Vandrilla for determination of
water content of I and II; P. Cunningham and D. Hora for technical
support; S. Painter for discussion and technical support; and Drs. T.
Baillie, M. Rowland, and K.C. Kwan for the critical review and
helpful discussions.
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of I to rats was ;91 to 100% of that after i.v. dosing of II at two lower
doses. The results indicate that I is a suitable prodrug that is effectively converted to II in vivo in the rat.
Likewise, the conversion of I to II in vivo was rapid in dogs such
that the level of I was not quantifiable 15 min post dosing of compound I at three doses studied. However, a nonproportional increase in
AUC values of II with dose was observed after i.v. administration of
I at 2 and 32 mg/kg, suggesting the elimination of II might have been
saturated at the high doses (Table 2). In comparison, pharmacokinetics of II appeared to be linear in dogs from 0.2 to 0.5 mg/kg. Deviation
from linear kinetics was observed at 2 mg/kg, in that the apparent
plasma clearance of II was decreased (from 2.3– 0.9 ml/min/kg). The
relative extent of exposure of II in plasma was less in dogs, ;59% at
the 0.5 mg/kg dose. Due to nonlinear kinetics observed in dogs at high
concentrations, the relative extent of exposure of II after i.v. dosing of
I at higher doses could not be determined. Taken all together, the
results suggest that I was effectively converted to II in vivo despite an
apparent saturation of the elimination of II.
Based on the results of in vitro stability of I, it is anticipated that the
conversion of I to II will be rapid in preclinical species (rat and dog) and
humans when I is administered i.v. As illustrated in this report, the
conversion of I to II in vivo was rapid in rats and dogs, therefore, it is
feasible to predict that the conversion of I to II will be rapid in humans
as well. This remains to be seen in clinical trails of I in the near future.
1373