Drug Metabolism Research (M.C., V.K.S., M.J.H., P.E.F., P.E.S,
J.J.V.) and
Structural, Analytical and Medicinal Chemistry
(D.A.K.), Pharmacia & Upjohn, Inc.
Delavirdine mesylate (U-90152T) is a highly specific nonnucleoside
HIV-1 reverse transcriptase inhibitor currently under development for
the treatment of AIDS. The metabolism of delavirdine was investigated in male and female cynomolgus monkeys after oral administration of
[14C-carboxamide]delavirdine mesylate at
single doses of 80 mg/kg and multiple doses of 160 to 300 mg/kg/day.
Desalkyl delavirdine was the major metabolite in circulation. In urine,
desalkyl delavirdine accounted for nearly half of the radioactivity,
with despyridinyl delavirdine and conjugates of desalkyl delavirdine
accounting for most of the remaining radioactivity. Bile was mostly
composed of desalkyl delavirdine and 6
-O-glucuronide
delavirdine, with parent drug, 4-O-glucuronide delavirdine,
and conjugates of desalkyl delavirdine as significant components. In
addition, several minor metabolites were observed in urine and bile of
delavirdine treated monkeys. The metabolism of delavirdine in the
monkey was extensive and involved N-desalkylation,
hydroxylation at the C-4 and C-6 positions of the pyridine ring,
hydroxylation at the C-4 position of the indole ring, pyridine
ring-cleavage, N-glucuronidation of the indole ring, and
amide bond cleavage as determined by MS and/or one-dimensional and
two-dimensional NMR spectroscopies. Phase II biotransformations
included glucuronidation, sulfation, and
-N-acetylglucosaminidation. The identification of the
N-linked -N-acetylglucosamine and
4-O-glucuronide metabolites of delavirdine represents novel
biotransformation pathways.
 |
Introduction |
Delavirdine mesylate (U-90152T,
fig. 1),
1-[(5-methanesulfonamido-1-H-indol-2-yl)-carbonyl]-4-[3-[(1-methylethyl)amino]-pyridinyl]piperazine monomethanesulfonate, is a potent and selective inhibitor of the HIV-11 reverse transcriptase (1, 2), an
enzyme which catalyzes the conversion of genomic viral RNA into
proviral DNA. The process of reverse transcription is essential to HIV
replication (3-5). Delavirdine is a member of the
bis(heteroaryl)piperazine class of nonnucleoside reverse transcriptase
inhibitors. Unlike nucleoside reverse transcriptase inhibitors
such as
3-azido-3-deoxythymidine (AZT, zidovudine) (6, 7),
2,3-dideoxyinosine (ddI, didanosine) (8-10), 2-3dideoxycytidine
(ddC, zalcitabine) (11), 2,3-didehydro-3-deoxythymidine (d4T,
stavudine) (12), and 2-deoxy-2-thiacytidine (3TC, lamivudine) (13,
14)which need to be converted by cellular enzymes to the active
deoxynucleotide triphosphates, delavirdine mesylate does not need
activation for inhibition of reverse transcriptase. Delavirdine has
been shown to selectively inhibit recombinant HIV-1 reverse
transcriptase with a median inhibitory concentration of 0.26 µM (2)
and block viral replication in peripheral blood lymphocytes of 25 primary HIV-1 isolates, including strains that were highly resistant to
AZT or ddI, with a 50% effective concentration of 0.066 µM (2). The
potent inhibition of replication by delavirdine was comparable to the
antiviral activity of nucleoside or other nonnucleoside reverse
transcriptase inhibitors (1, 2). Unlike nucleoside reverse
transcriptase inhibitors that exhibit serious toxicity (15, 16),
delavirdine has low cellular toxicity, causing less than 8% reduction
in peripheral blood lymphocyte viability at 100 µM, and exhibits no
significant inhibition of cellular polymerases 
and , with a
median inhibitory concentration of >440 µM (2). Delavirdine mesylate
is currently under phase III clinical evaluations. In
the course of the preclinical evaluation of delavirdine, its metabolism
was investigated in male and female cynomolgus monkeys after single and
multiple oral dose administration of
[14C-carboxamide]delavirdine mesylate. The results from
this study, including the identification of the metabolites of
delavirdine, are presented.
Materials and Methods
Chemicals.
All chemicals used in this study were of analytical grade. Solvents
were Burdick & Jackson high purity grade (Burdick & Jackson, Muskegon,
MI). Water was distilled and purified through a Milli-Q reagent water
system (Millipore Corp., Bedford, MA). Ultima Gold (Packard Instrument
Co., Meriden, CT) was used for liquid scintillation counting (LSC).
Carbo-sorb E (Packard) and Permafluor E+ (Packard) were
used for combustion of samples. Ultima-Flo M (Packard) was used as
scintillant for flow-through detection. -Glucuronidase (EC 3.2.1.31
from Helix pomatia, Type H-5), sulfatase (EC 3.1.6.1 from
Aerobacter aerogenes, Type VI), and Trizma buffer (Tris-HCl 0.2 M, pH 7.4) were obtained from Sigma Chemical Company (St. Louis,
MO). Methanol-d4 (99.96% D) was purchased from Cambridge Isotope Laboratories (Cambridge, MA).
Delavirdine mesylate, [14C-carboxamide]delavirdine
mesylate, U-96183, U-102466, U-88703, and U-96364 were synthesized by
Pharmacia & Upjohn, Inc. The specific activity of the radiolabeled test substance was 34.7 µCi/mg, with a radiochemical purity of >99% by
HPLC. The chemical purity of the nonradiolabeled delavirdine mesylate
was 97.6%. The structure and position of the 14C label are
shown in fig. 1.
Animals.
Cynomolgus monkeys were obtained from the Pharmacia & Upjohn primate
colony. The body weight of the monkeys ranged from 4.4 to 6.2 kg.
Monkeys were housed in individual stainless steel metabolism cages.
Environmental conditions were maintained as follows: lights 12 hr on/12
hr off, relative humidity 40-60%, temperature 68-75°F, ventilation
18-20 room air changes per hr. Monkeys were provided with food
(Certified Monkey Diet 5048, PMI Feeds Inc., St Louis, MO, supplemented
with fresh fruit) and water ad libitum. Monkeys were fasted
overnight prior to and 4 hr after radiolabeled dose administration. The
animals in this study were cared for and used in accordance with
The Guide for the Care and Use of Laboratory Animals, DHEW
Publication (National Institutes of Health) 85-23, 1985 and with
The Animal Welfare Act Regulation 9 CFR 3, August 15, 1989 and as modified on March 19, 1991.
Instrumentation.
A Packard Tri-Carb Liquid Scintillation Analyzer Model 1900 CA or Model
1900TR (Packard Instrument Co.) was used for radioactivity counting. A
TurboVap LV evaporator (Zymark Corporation, Hopkinton, MA) or a
SpeedVac concentrator Model SVC 100H or Model AS260 (Savant Instruments, Inc., Farmingdale, NY) was used to concentrate samples.
Chromatography.
The HPLC system consisted of a Perkin Elmer Series 410 quaternary pump
(Perkin-Elmer Corp., Norwalk, CT), a Perkin Elmer ISS 200 LC sample
processor, a Perkin Elmer LC-235C diode array detector, and a
Radiomatic Model A-525 flow-through detector (Packard Instrument Co.,
Meriden, CT) containing a 500-µl liquid cell. A Foxy 200 fraction
collector (Isco, Inc., Lincoln, NE) equipped with a diverter valve was
used to collect fractions at 0.25- to 1-min intervals on a recycling
mode.
Solid phase extraction (SPE) was performed using Bakerbond octadecyl
extraction columns (J.T. Baker, Inc., Phillipsburg, NJ) containing 500 mg of C18 packing. Columns were washed with approximately 3 ml acetonitrile, then equilibrated with 6 ml 0.1 M ammonium acetate
buffer (pH 4) before use. A Supelco Visiprep vacuum manifold (Supelco,
Inc., Bellefonte, PA) was used for SPE.
Mass Spectrometry.
Particle beam-liquid chromatography-mass spectrometry (PB-LC-MS) was
performed using a Finnigan 4021 quadrupole (Finnigan MAT, San Jose, CA)
or a VG Autospec Q hybrid (Fisons, Manchester, UK) mass spectrometer
equipped with a particle beam interface (Thermabeam, Extrel Corp.,
Pittsburgh, PA; interface built at Pharmacia & Upjohn, Inc.). The
instrument was operated in the positive chemical ionization (CI) mode
using ammonia as reagent gas. Electron energy was set at 70 eV.
Interface temperatures were controlled by Vestec thermospray
electronics (Vestec, Houston, TX). Data were acquired and reduced by a
Vector II data system (Teknivent Corporation, Maryland, MO). HPLC
analyses were performed on a system consisting of a Perkin Elmer ISS
100 sample processor (Perkin-Elmer Corp., Norwalk, CT) interfaced to a
Perkin Elmer Series 410 quaternary pump with a Perkin Elmer SEC-4
solvent environment control and a Waters 490MS UV detector (Waters,
Milford, MA). Samples were analyzed on a YMC 5µ-basic 4.6 mm i.d. × 25 cm column (YMC, Inc., Wilmington, NC) connected to a YMC 5µ-basic
4.0 mm i.d. × 2.3 cm guard cartridge. The mobile phase consisted of
elution at 0.5 ml/min with a 20-min linear gradient from 10% A/90% D
to 60% A/40% D, followed by 20 min 60% A/40% D, then a 10-min
linear gradient to 80% A/20% D where A = acetonitrile, D = 0.1 M ammonium acetate (pH 4).
Electrospray ionization (ESI) and atmospheric pressure chemical
ionization (APCI) mass spectra were obtained on a Finnigan-MAT TSQ 7000 triple quadrupole mass spectrometer (Finnigan-MAT, San Jose, CA)
directly coupled to the HPLC system via a Finnigan
atmospheric pressure ionization (API) source. Data were collected on a
DEC 3000 Model 300X computer running OSF/1 version 2.0 as the operating system. The mass spectrometer was controlled using Instrument Control
Language version 8.0 and the data were processed using Interactive
Chemical Information System version 8.1.1 software. A tuning solution
containing 1 mg/ml delavirdine mesylate was infused at a rate of 5 µl/min by a syringe pump into the mobile phase flow path via a T
connector. Argon was used as collision gas at a pressure of 1 × 10-6 mbarr and collision energy of 50 eV. Samples were
analyzed on a YMC 5µ-phenyl 4.6 mm i.d. × 25 cm column connected to
a YMC 5µ-phenyl 4.6 mm i.d. × 2.3 cm guard cartridge. The mobile
phase consisted of elution at 0.5 ml/min with a 25 min 20% A/80% D, 5-min linear gradient to 80% A/20% D, followed by 10 min 80% A/20% D where A = acetonitrile, D = 0.1 M ammonium acetate (pH 4).
Nuclear Magnetic Resonance Spectroscopy.
1H NMR spectra were recorded at 500 MHz using a Bruker
AMX-500 spectrometer (Bruker Instruments Inc., Billerica, MA). Data were processed on a Bruker X-32 computer using Bruker UXNMR software version 930901.3. Samples were dissolved in approximately 300-500 µl
methanol-d4 in a 3 mm i.d. Teflon insert (WILMAD, Buena,
NJ); the Teflon insert was placed in a 5 mm NMR tube (WILMAD, Buena, NJ). Spectra were recorded at a temperature of 300°K as free
induction decays of 32K complex points. A resolution enhancing Gaussian window was applied to the free induction decays of selected samples to
better resolve the couplings of the sugar resonances. The residual CHD2OH pentet was used as reference at 3.30 ppm.
2D NMR experiments were recorded in the phase-sensitive mode using the
Time Proportional Phase Increment method (17) to obtain quadrature
detection in the second dimension. All experiments used presaturation
of the large solvent hydroxyl at 4.82 ppm during a relaxation delay of
0.8-1.0 sec. Data tables were either 1K by 1K or 2K by 1K with 256 or
512 increments in F1 and 1K or 2K complex free induction decay data
points in F2. 1H-1H double quantum filter COSY
spectra (18) used sinebell-squared window functions shifted by 
/3
radians. All other 2D experiments used sinebell-squared window
functions which were shifted by /2 radians. The ROESY spectrum (19)
used a 2,070 Hz continuous-wave spin-locking field applied for 200 msec
at the frequency of the solvent hydroxyl peak. TOCSY spectra (20) used
a 60 msec spin-lock. Proton-carbon HMQC (21) experiments used a delay
of D2 = 1/(2JHC) = 3.2 msec and globally optimized
alternating-phase rectangular pulses decoupling (22) of 13C
during acquisition. 2D spectra were referenced to the solvent at 3.30 (1H) and 49.0 ppm (13C).
Ultraviolet Spectrometry.
UV spectra were obtained on a Perkin Elmer LC-235C diode array
detector. Effluent was monitored at 295 and 255 nm, using a 5-nm band
width, 16-sec peak width, and a scanning rate of 1 scan every 5 sec.
Peak purity information was obtained by comparing the absorbance of
each and every defined wavelength in 5-nm increments of the UV spectrum
taken on the leading edge with the corresponding absorbance of the
spectrum taken on the trailing edge (23). This comparison was
determined at approximately 20% peak height. A given peak was taken as
homogeneous if the peak purity index was 1.5 or lower.
Dosing and Sample Collection.
Delavirdine mesylate and [14C-carboxamide]delavirdine
mesylate were dissolved in 80% propylene glycol/20% water containing
3 µl methanesulfonic acid per ml to a final concentration of 150 mg/ml. In addition, a nonradiolabeled formulation was prepared at a
concentration of 150 mg/ml. Two male and two female monkeys received
single oral radiolabeled doses of approximately 80 mg/kg and 47 µCi/kg. One additional male and one female monkey were administered
multiple oral nonradiolabeled doses of approximately 160 mg/kg/day
given twice a day for 7 days (one morning dose and a second dose 8 hr
later), followed on day 8 by a single oral radiolabeled dose of
approximately 80 mg/kg and 47 µCi/kg. Doses were measured
gravimetrically and administered by oral gavage using a dosing syringe
attached to an intubation tube. Immediately after radiolabeled dose
administration, monkeys received approximately 5 ml 0.01 N HCl to
ensure complete transfer of the dosing formulation. Urine was collected
from the monkeys over the time periods 0-12, 12-24, and at 24-hr
intervals up to 168 hr after radiolabeled dosing. Urine was immediately
analyzed or stored at 
20°C until further analysis. Blood samples
(3-ml aliquots, total blood withdrawn 33 ml for single-dose and 42 ml
for multiple-dose studies) were collected at scheduled times through
the 48-hr period after the radiolabeled dose, stored on ice, and
centrifuged to separate the plasma.
Terminal bile was obtained at the time of sacrifice, 10-13 hr after
the last dose, from a male monkey given 240 mg/kg/day and a female
monkey administered 300 mg/kg/day doses of delavirdine mesylate (doses
given three times a day) from a 3-month toxicity study.
Sample Preparation and Total Radiocarbon Analysis.
Aliquots of urine containing 500 µl (3 replicates) and 100 µl
portions of plasma (2 replicates) were measured gravimetrically, mixed
with 5 ml of Ultima Gold, and analyzed by LSC. Radioactivity was
measured by LSC with 1-10 min counting. Count rates in cpm were
corrected to dpm using quench curves generated from sealed quenched
standards in either Ultima Gold cocktail. The limit of detection was
~40 dpm (2x background).
HPLC Profiles of Plasma and Urine Samples.
Plasma and urine samples were profiled for parent drug and metabolites
by HPLC with flow-through radiochemical detection. Chromatographic
analyses were performed on a YMC 5µ-basic 4.6 mm i.d. × 25 cm column
using gradient elution at 1.0 ml/min with 10% A/90% D for 5 min,
followed by a 35-min linear gradient to 60% A/40% D, then 60% A/40%
D for 5 min (where A = acetonitrile, D = 0.1 M ammonium
acetate buffer (pH 4)). The HPLC effluent was mixed with Ultima-Flo M
in a 1:3 ratio. Peaks of radioactivity were quantified on the
Radiomatic detector using peak area integration. Column recovery was
determined by comparing the total radioactivity from the sum of the
integrated peaks in the 14C chromatogram with total
radioactivity. The radioactivity in a given chromatographic peak was
expressed as a percentage of the total radioactivity eluted during the
analysis. Values were not corrected for column recovery since
essentially all of the injected radioactivity was recovered from the
column. The amounts of delavirdine and its metabolites in urine were
expressed as percentages of the radioactivity in urine.
A portion of plasma ranging in volume from 100 to 200 µl was mixed
with 2 volumes of acetonitrile followed by centrifugation at 14,000 rpm
for 5 min at 4°C. The supernatant was evaporated to dryness and
reconstituted in 100 µl 10% acetonitrile/90% 0.1 M ammonium acetate
buffer (pH 4). A 75-µl aliquot of prepared plasma sample was analyzed
by HPLC with flow-through radiochemical detection.
Urine samples were centrifuged to remove large particulates. A 200-µl
portion of urine samples collected over the time periods 0-12, 12-24,
24-48, and 48-72 hr was directly analyzed by HPLC with flow-through
radiochemical detection using gradient elution (vide supra).
HPLC Profiles of Bile Samples.
Bile samples were directly profiled for parent drug and metabolites by
HPLC with UV detection at 295 nm. Chromatographic conditions were the
same as described above. Peaks were quantified using peak area
integration assuming the same molar absorptivity. Peaks were assigned
as drug-related material based on chromatographic comparison of bile
from delavirdine treated and control animals.
Metabolite Isolation.
Metabolites were isolated from appropriate matrices as described below.
In general, at each purification step an aliquot of each fraction was
analyzed by LSC; from these data aliquots of selected fractions were
analyzed by HPLC with flow-through radiochemical detection. Fractions
containing the same metabolite(s) were combined, weighed, and an
aliquot was gravimetrically measured and analyzed by LSC. The amount of
metabolite(s) in the fraction was estimated from the specific activity
of the dosing formulation. Purification of metabolites from bile was
monitored by HPLC with UV detection.
MET-4 was isolated as follows: Urine (20 ml) collected over the time
period 0-12 hr from a female monkey given a 80 mg/kg single oral dose
of [14C-carboxamide]delavirdine mesylate was lyophilized.
The residue was dissolved in approximately 5 ml 10% acetonitrile/90%
0.1 M ammonium acetate (pH 4) to afford 15.7 × 106
dpm, 11.4 mg of drug-related material. A portion of the concentrated urine was purified by HPLC with gradient elution (YMC 5µ-basic 10 mm
i.d. × 25 cm, 5 injections of 800 µl each, 3.0 ml/min, 5-min 10%
A/90% D, 35-min linear gradient to 60% A/40% D, 5-min 60% A/40% D,
A = acetonitrile, D = 0.1 M ammonium acetate buffer (pH 4))
to give 1.8 mg (2.48 × 106 dpm) of partially purified
MET-4. The partially purified MET-4 was concentrated (2.45 × 106 dpm, 1.8 mg of drug-related material) and further
resolved into four metabolites by purification on a phenyl-bonded
column (YMC 5µ-phenyl 4.6 mm i.d. × 25 cm, 10 injections of 200 µl
each, 1.0 ml/min, 25 min 15% A/85% D, 10-min linear gradient to 60%
A/40% D, 10 min 60% A/40% D; A = acetonitrile, D = 0.1 M
ammonium acetate (pH 4)). After lyophilization, four purified
metabolites were obtained: 0.17 mg of MET-4a (0.23 × 106 dpm), 0.22 mg of MET-4b (0.30 × 106
dpm), 0.41 mg of MET-4c (0.56 × 106 dpm), and 0.06 mg
of MET-4d (0.08 × 106 dpm).
MET-16 was isolated as follows: Terminal bile (1.0 ml) collected from a
male monkey given 240 mg/kg/day doses of delavirdine mesylate for 3 months was purified by C18 SPE. Two SPE columns were
stepwise eluted with 5 ml each 0%, 10%, 35%, and 90% acetonitrile in 0.1 M ammonium acetate buffer (pH 4). The 10% acetonitrile fractions were combined and purified by semi-preparative HPLC with
gradient elution (YMC 5µ-basic 10 mm i.d. × 25 cm, 50 injections of
200 µl each, 3.0 ml/min, 5-min 10% A/90% D, 35-min linear gradient to 60% A/40% D, 5-min 60% A/40% D, A = acetonitrile, D = 0.1 M ammonium acetate buffer (pH 4)). Fractions containing the peak eluting at approximately 23 min were combined, concentrated, and further purified by isocratic reversed phase HPLC (YMC 5µ-basic 4.6 mm i.d. × 25 cm, 52 injections, 6.6 ml total, 15% acetonitrile/15% methanol/70% 0.1 M ammonium acetate buffer (pH 4)). Fractions containing the peak eluting at 17.6 min were combined and concentrated to afford the purified MET-16.
Metabolite Identification.
Portions of plasma (200 µl) collected 3 and 4 hr post dose from a
female monkey given a 80 mg/kg single-dose of
[14C-carboxamide]delavirdine mesylate were combined and
mixed with 400 µl acetonitrile. After centrifugation at 14,000 rpm
for 5 min at 4°C, the supernatants were combined, concentrated to
dryness, and reconstituted in 225 µl 15% acetonitrile/90% 0.1 M
ammonium acetate buffer (pH 4). An aliquot of the concentrated plasma
sample was analyzed by LC-ESI-MS.
A 5-ml portion of urine collected over the time period 0-12 hr from a
female monkey given a 80 mg/kg single-dose of
[14C-carboxamide]delavirdine mesylate was concentrated to
dryness and reconstituted in approximately 1.2 ml 15%
acetonitrile/85% 0.1 M ammonium acetate buffer (pH 4). An aliquot of
the concentrated urine sample was analyzed by LC-APCI-MS and
LCESI-MS.
Terminal bile collected from a male monkey given 240 mg/kg/day doses of
delavirdine mesylate for 3 months was directly analyzed by LC-ESI-MS.
The purified MET-4a was dissolved in methanol for ESI-MS analysis. The
purified MET-4b was dissolved in 300-400 µl of
methanol-d4 for NMR analysis. A 50-µl aliquot of MET-4b
in methanol-d4 was evaporated to dryness and reconstituted
in 100 µl methanol for ESI-MS analysis. The purified MET-4c was
dissolved in 500 µl methanol. A 20-µl aliquot was diluted with 180 µl 0.1 M ammonium acetate buffer (pH 4) for PB-LC-MS and ESI-MS
analyses. The remaining sample was evaporated to dryness and processed
as described above for NMR analysis. The purified MET-4d was dissolved
in 100 µl methanol for ESI-MS analysis.
A portion of the purified MET-16 was dissolved in methanol for ESI-MS
analysis. The remaining sample was dissolved in methanol-d4 as described above for NMR analysis.
Enzyme Hydrolysis.
A 2.4-ml portion of urine collected 0-12 hr post-dose from a female
monkey given a 80 mg/kg single-dose of
[14C-carboxamide]delavirdine mesylate was lyophilized and
the residue reconstituted in 2.4 ml of Trizma buffer. A 600-µl
aliquot of the sample was mixed with 600 µl of sulfatase solution (2 units/ml in Trizma buffer) and incubated at 37°C for 1 hr. A 100-µl
aliquot of the solution was analyzed by HPLC with radiochemical
detection. The HPLC conditions consisted of 1.0 ml/min elution on a YMC
5µ-phenyl 4.6 mm i.d. × 25 cm column with 25-min 15% A/85% D,
5-min linear gradient to 60% A/40% D, 10-min 60% A/40% D (A = acetonitrile, D = 0.1 M ammonium acetate buffer (pH 4)). Samples
were also analyzed on a YMC 5µ-basic 4.6 mm i.d. × 25 cm column
eluted at 1.0 ml/min with 5-min 10% A/90% D, 35-min linear gradient
to 60% A/40% D, 5-min 60% A/40% D. A control sample containing
monkey urine and Trizma buffer was carried out similarly.
A 25-µl portion of terminal bile collected from a male monkey given
240 mg/kg/day doses of delavirdine mesylate in the 3-month toxicity
study was mixed with 200 µl 0.1 M sodium acetate (pH 5) and 200 µl
-glucuronidase solution (45,500 units/ml in 0.2% saline). The
mixture was incubated at 37°C for 1 hr. A 25-µl aliquot of the
sample was analyzed by gradient HPLC with UV detection at 295 nm. A
control sample was prepared by mixing 25 µl of bile with 200 µl 0.1 M sodium acetate (pH 5) and 200 µl 0.2% saline and analyzed
similarly.
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Results |
Plasma Metabolite Profiles.
A summary of the analyses of plasma samples for delavirdine and its
metabolites by HPLC with radiochemical detection is presented in table
1. Desalkyl delavirdine was the major radioactive
component in circulation after single dose and in male monkeys after
multiple dose, constituting 58% and 54% of the radioactivity in 0-8
hr plasma, respectively, with delavirdine accounting for 20% and 31%.
In female monkeys given multiple doses, delavirdine and desalkyl delavirdine were the major radioactive components in circulation. The
remaining radioactivity in plasma was associated with MET-4 and MET-6.
A representative chromatogram is shown in fig. 2.
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TABLE 1
Metabolite profiles of plasma and urine samples after single and
multiple oral dose administration of delavirdine
mesylatea
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Fig. 2.
Representative HPLC chromatograms of monkey
plasma, urine, and bile after multiple-dose administration of
delavirdine mesylate.
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HPLC Profiles of Urine.
Urine was analyzed for delavirdine and its metabolites by HPLC with
flow-through radiochemical detection (table 1). The metabolism of
delavirdine was extensive in the monkey, with no detectable amount
excreted in urine as intact drug. After single and multiple doses, the
major component in urine was MET-5, accounting for 40-56% of the
radioactivity in urine. In addition, 19-29% of the radioactivity in
urine was excreted as MET-4 and 9-18% as MET-2. Several minor
metabolites were also observed in urine, including MET-10, MET-3, and
MET-6. A representative chromatogram is shown in fig. 2.
HPLC Profiles of Bile.
Bile was analyzed for delavirdine and its metabolites by HPLC with UV
detection. Extensive metabolism was observed in bile samples from both
male and female monkeys. The major biliary components (% of
drug-related material in bile) were desalkyl delavirdine (20-31%) and
MET-6 (19-27%). Parent drug constituted 11%. Several minor
metabolites were also observed in bile, including MET-2 (4%), MET-3
(7-11%), MET-4 (11-15%), MET-16 (5-9%), and MET-7 (4-8%). A
representative chromatogram is shown in fig. 2.
Metabolite Isolation and Identification.
The presence of delavirdine in plasma, bile, and urine was indicated by
HPLC retention time and UV spectrum comparisons with a synthetic
standard and was confirmed by LC-MS analysis. The ESI mass spectrum
showed a pseudomolecular ion (MH+) for parent drug at
m/z 457 (base peak) (fig. 3, table
2). Structurally informative fragment ions at
m/z 378, 247, and 221 arise from loss of
CH3SO2, cleavage of the carbonyl-indole
linkage, and cleavage of the carbonyl-piperazine bond, respectively.
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Fig. 3.
Mass spectra of delavirdine and its
metabolites.
Top left, ESI mass spectrum of delavirdine from monkey plasma; bottom
left, ESI mass spectrum of MET-5 from monkey plasma; top right, ESI
product-ion mass spectrum of MET-6 from monkey urine; bottom right, ESI
product-ion mass spectrum of MET-16 from monkey urine.
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The major component in monkey plasma, bile, and urine, MET-5, had the
same HPLC retention time and UV spectrum as a synthetic standard of
desalkyl delavirdine (U-96183). The presence of this metabolite in
plasma, bile, and urine was confirmed by LC-MS. The ESI mass spectrum
of MET-5 showed a pseudomolecular ion at m/z 415, 42 amu
lower than parent drug, indicative of loss of the isopropyl side chain
(fig. 3, table 2). Loss of CH3SO2 gave an ion
at m/z 336. Cleavage of the carbonyl-piperazine bond gave ions at m/z 179 and 237, while cleavage of the
carbonyl-indole linkage resulted in a fragment at m/z 205. Further confirmation of the presence of desalkyl delavirdine in monkey
urine was obtained by PB-LC-CI-MS.
MET-3 was present as a minor component in bile and urine of
delavirdine-treated monkeys. Elution of MET-3 as a broad peak indicated
the presence of more than one metabolite. MET-3 remained unchanged upon
treatment of bile with -glucuronidase (containing -glucuronidase
and sulfatase activities), suggesting that this metabolite was neither
a -glucuronic acid nor sulfate conjugate. MET-3 was unstable and on
heating degraded to desalkyl delavirdine. This metabolite was not
further characterized.
MET-2 was observed as a significant metabolite in urine and as a minor
component in bile after administration of delavirdine mesylate. This
metabolite had similar retention time as a synthetic standard of
despyridinyl delavirdine (U-102466). Identification of MET-2 as
despyridinyl delavirdine was established by HPLC co-injection with a
synthetic standard and was confirmed by LC-MS. The ESI mass spectrum of
MET-2 showed a pseudomolecular ion at m/z 323, indicating
cleavage of the pyridine ring and an ion at m/z 237 resulting from cleavage of the carbonyl-piperazine linkage.
Another major metabolite in bile, MET-6, was also observed as a minor
metabolite in plasma and urine of delavirdine-treated monkeys.
Treatment of bile with -glucuronidase (containing glucuronidase and
sulfatase activities) resulted in the disappearance of MET-6 concomitant with an increase in MET-7. On further standing at room
temperature within a few hours, MET-7 degraded to despyridinyl delavirdine (MET-2). Upon treatment of monkey bile with sulfatase (containing no detectable -glucuronidase activity), MET-6 remained unchanged. These data suggested that MET-6 was a glucuronic acid conjugate of MET-7.
Confirmation of MET-6 as a glucuronide was obtained by LC-MS analyses
of monkey plasma, bile, and urine, and by LC-MS-MS of bile. The ESI
mass spectrum of MET-6 showed a pseudomolecular ion at m/z
649, 192 amu higher than parent drug, indicative of addition of a
hydroxy group followed by conjugation with glucuronic acid (fig. 3,
table 2). Loss of the glucuronic acid moiety gave the base peak at
m/z 473. Subsequent cleavage of the carbonyl-piperazine bond
and the carbonyl-indole linkage gave ions at m/z 237 and 263, respectively, and indicated substitution on the right hand side of
the molecule (i.e., piperazine ring, pyridine ring, or isopropyl group). Loss of the isopropyl moiety from the ion at m/z 263 to generate an ion at m/z 221 indicated
that the substituent was located on the pyridine or piperazine rings.
The position of substitution was determined to be at the pyridine ring
C-6 by HPLC retention time comparison to MET-6 isolated from rat bile and identified by 1H NMR and MS (24).
Confirmation of MET-7 as 6-hydroxy delavirdine was obtained by LC-MS
analysis of monkey urine. The APCI and ESI mass spectra of MET-7 showed
a pseudomolecular ion at m/z 473 (table 2), 16 amu higher
than parent drug and indicative of the presence of a hydroxyl group.
Cleavage of the carbonyl-indole linkage gave an ion at m/z
263, 16 amu higher than the corresponding ion for parent drug, and
indicated substitution on the pyridine or piperazine rings. These data
together with the fact that enzyme hydrolysis of
6-O-glucuronide delavirdine (MET-6) generated MET-7,
identified MET-7 as 6-hydroxy delavirdine.
MET-10 and MET-12 were observed as minor urinary metabolites using
radiochemical and UV detectors, respectively. These metabolites eluted
at the same HPLC retention times as the synthetic standards U-96364
(indole carboxylic acid metabolite) and U-88703
(N-isopropylpyridinepiperazine metabolite), respectively,
arising from cleavage of the amide bond. Confirmation of the presence
of the indole carboxylic acid metabolite in monkey urine was pursued by
LC-MS. However, no spectroscopic information could be obtained because
of the small amount present and to the insensitivity of the technique
for this metabolite. On the other hand, ESI-MS was more sensitive for
the N-isopropylpyridinepiperazine metabolite and confirmed a
pseudomolecular ion at m/z 221 (table 2).
MET-4 was observed as a significant component in urine of delavirdine
treated monkeys. Phenyl-phase chromatography indicated that four
metabolites, MET-4a, MET-4b, MET-4c, and MET-4d, were present.
Incubation of monkey urine with sulfatase resulted in the disappearance
of MET-4b and MET-4d, concomitant with an increase in MET-15b and
MET-15d, while MET-4a and MET-4d remained unchanged. These data
indicated that MET-4b and MET-4c contained a phenolsulfate group, while
MET-4a and MET-4c did not. To identify these metabolites, isolation
from monkey urine was pursued.
MET-4a showed a UV 
max at 296 nm, indicating the
presence of an indole ring. HPLC retention time comparison showed that
the monkey MET-4a was identical to the previously isolated dog urinary metabolite (25) which had been identified as the sulfamate conjugate of
desalkyl delavirdine. Analysis of monkey urine by APCI-MS (fig. 4, table 2) confirmed this assignment with a
pseudomolecular ion at m/z 495, 80 amu higher than desalkyl
delavirdine, indicative of sulfamate conjugation. Loss of
CH3 resulted in an ion at m/z 480. Loss of
SO3H gave an ion at m/z 415, and subsequent
cleavage of the carbonyl-piperazine linkage resulted in ions at
m/z 179 and 237, while cleavage of the carbonyl-indole bond
gave an ion at m/z 207. Loss of
CH3SO2 from the ion at m/z 237 resulted in an ion at m/z 159.
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Fig. 4.
Mass spectra of delavirdine and its
metabolites from monkey urine.
Top left, APCI mass spectrum of MET-4a; bottom left, ESI mass spectrum
of MET-4b; top right, APCI mass spectrum of MET-4c; bottom right, ESI
mass spectrum of MET-4c.
|
|
The purified MET-4b showed a UV max at 301 nm,
suggesting the presence of an indole ring. Diode array analysis gave a
purity index of 1.4 and indicated the absence of UV absorbing
impurities. The 1H NMR (500 MHz, methanol-d4,
fig. 5, table 3) spectrum showed the
presence of six aromatic protons, eight piperazine protons, and a
methylsulfonate group. Four aromatic resonances were similar to
corresponding resonances in the parent drug and were assigned to
protons on the indole ring (
7.55, 7.42, 7.17, and 6.83 ppm). A
resonance for the pyridine H-6 was not observed and the remaining two
aromatic resonances for the pyridine ring protons H-4 and H-5 at 7.13 and 6.91 ppm, respectively, were shifted upfield compared with
corresponding resonances in the parent drug ( 7.50 and 7.37 ppm). In
addition, the H-4 and H-5 protons in MET-4b appeared as doublets with
a coupling constant J4
-5 of 8.3 Hz, whereas in the parent
drug these protons were observed as doublets of doublets with coupling
constants J4-5 of 8.3 Hz, J4-6 of 1.4 Hz,
and J5-6 of 5.7 Hz.
These data established that MET-4b was substituted at the pyridine ring
C-6. Assignments were confirmed by 2D COSY NMR (table
4), which established homonuclear spin coupling between H-7 and H-6, as well as between H-4 and H-5. A 2D TOCSY NMR experiment further confirmed the assignments, with cross peaks between
H-4 and H-7, H-7 and H-6, H-4 and H-6, and H-4 and H-5. Additional
resonances in the 1H NMR spectrum of MET-4b corresponded to
the piperazine ring protons ( 3.99 and 3.19 ppm) and the
methylsulfonate group ( 2.88 ppm). Resonances for the isopropyl side
chain were not observed and indicated N-desalkylation had
occurred. The 6-substitution pattern was confirmed with a 2D
heteronuclear correlation experiment (table 4), which showed four
13C aromatic resonances for the indole ring carbons ( 121, 116, 106, and 113 ppm) and two resonances for the pyridine ring
carbons at 121 and 111 ppm, corresponding to C-4 and C-5,
respectively. The resonance for C-5 was shifted upfield relative to
the corresponding resonance in the parent drug ( 121 ppm), while
C-4 remained unchanged. These spectroscopic data together with the
enzyme hydrolysis results indicated a desalkyl delavirdine nucleus
substituted at C-6 with a moiety containing an exchangeable proton,
such as a sulfate group. Confirmation of MET-4b as 6-sulfate desalkyl delavirdine was obtained by ESI and APCI mass spectrometries (fig. 4,
table 2), which showed a pseudomolecular ion at m/z 511, 96 amu higher than desalkyl delavirdine and indicative of addition of a
sulfate group. Loss of sulfate gave an ion at m/z 431, subsequent cleavage of the carbonyl-indole bond gave an ion at
m/z 221. Cleavage of the carbonyl-piperazine linkage
resulted in ions at m/z 275 and 237.
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Fig. 5.
1H NMR spectra (500 Mhz,
methanol-d4) of the aromatic region of
delavirdine and its metabolites.
From top to bottom: delavirdine, MET-4b, MET-4c, and MET-16.
Resonances for the unhydrolyzed MET-4c (bold) and MET-5 (normal) resulting from hydrolysis of MET-4c are included.
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|
MET-4c showed a UV max at 300 nm and suggested the
presence of an indole ring. The isolated MET-4c was extremely unstable and on concentration at room temperature hydrolyzed partially to give a
radioactive peak with similar retention time as desalkyl delavirdine.
These data suggested that MET-4c was a conjugate of desalkyl
delavirdine.
The CI mass spectrum of MET-4c (table 2) showed a pseudomolecular ion
at m/z 618, 203 amu higher than desalkyl delavirdine. The
odd molecular weight (MW 617) for MET-4c indicated an odd number of
nitrogens (25). Loss of acetate gave an ion at m/z 576, successive losses of water gave ions at m/z 558, 540, and 522. Loss of CH3SO2 from m/z 576 resulted in an ion at m/z 498. Consecutive loss of water
from m/z 618 gave ions at m/z 600, 582, and 564. The data suggested the presence of a sugar moiety containing an acetyl
group. Cleavage of the sugar moiety gave ions at m/z 415 and
219. Loss of CH3SO2 from m/z 415 gave an ion at m/z 337, subsequent cleavage of the
carbonyl-indole linkage gave an ion at m/z 133. Cleavage of
the carbonyl-piperazine bond gave a fragment at m/z 179, while cleavage of the carbonyl-indole linkage resulted in an ion at
m/z 207. Cleavage of the piperazine-pyridine linkage gave
ions at m/z 323 and 95. These spectroscopic data indicated that biotransformation had occurred on the amino group of the pyridine
ring.
Although ions from m/z 522 to 618 observed by CI-MS
represented <0.2% of the total ion current, the reconstructed mass
chromatograms indicated that these ions were not noise signals. This
was confirmed by ESI-MS, with a base peak at m/z 618 corresponding to the pseudomolecular ion and an ion at m/z
415 arising from cleavage of the sugar moiety. Likewise, the APCI mass
spectrum of MET-4c showed a pseudomolecular ion at m/z 618 (fig. 4, table 2). Loss of water gave an ion at m/z 600. Loss of CH3SO2 and acetate gave an ion at
m/z 498. Cleavage of the sugar gave an ion at m/z
415, and subsequent cleavage of the carbonyl-piperazine linkage gave
ions at m/z 179 and 237. Loss of
CH3SO2 from m/z 237 resulted in an
ion at m/z 159. Exact mass measurements gave a molecular
mass of 618.2359 (calc. 618.2346) and an empirical formula of
C27H36N7O8S1.
The 1H NMR (500 MHz, methanol-d4, fig. 5, table
3) spectrum of MET-4c was very complex because of the presence of
unhydrolyzed MET-4c and desalkyl delavirdine and the acetylated sugar
arising from hydrolysis of MET-4c. However, because of the twofold
ratio of MET-4c relative to desalkyl delavirdine, the resonances
corresponding to unhydrolyzed MET-4c could be discriminated from those
of desalkyl delavirdine and the acetylated sugar. MET-4c showed the
presence of seven aromatic protons, eight piperazine protons, a
methylsulfonate group, an N-acetyl group, and protons
corresponding to a sugar moiety. Four aromatic resonances were similar
to corresponding resonances in the parent drug and were assigned to
protons on the indole ring ( 7.56, 7.43, 7.17, and 6.85 ppm). Three
aromatic resonances corresponded to the pyridine ring protons H-4,
H-5, and H-6 ( 7.17, 7.01, and 7.68 ppm, respectively).
Assignments were confirmed with a 2D COSY NMR experiment (table 4),
which established homonuclear spin coupling between H-5 and H-6, H-6 and H-7, and H-4 and H-5. Likewise, a 2D TOCSY NMR experiment showed
cross peaks between H-4 and H-6, H-5 and H-6, H-4 and H-7, H-4 and
H-6, H-6 and H-7, and H-4 and H-5. The remaining resonances in the
aromatic region that integrated for approximately half of the protons
for MET-4c were assigned to desalkyl delavirdine (table 3). Additional
resonances in the 1H NMR spectrum of MET-4c corresponded to
the piperazine ring protons ( 4.07 and 3.03 ppm), the
methylsulfonate group ( 2.89 ppm), the N-acetyl group
( 2.01 ppm), and the sugar protons ( 5.91, 4.55, 4.01, 3.87, 3.67, 3.55, and 3.44 ppm). Resonances for the isopropyl side chain were
not observed and indicated N-desalkylation had occurred. A
coupling constant between the anomeric H-1" ( 5.91 ppm) and H-2"
( 4.55 ppm) of 7.3 Hz confirmed the -linkage of the sugar. Sugar
resonances were assigned with 2D COSY (fig. 6) and 2D
TOCSY NMR experiments. In addition, 1H homonuclear
decoupling experiments showed coupling constants J2"-3",
J3"-4", and J4"-5" of 9.3, 10 and 8.5 Hz,
respectively, and together with a 2D NOESY experiment established axial
conformations for H-2", H-3", H-4", and H-5" and identified MET-4c as
-N-acetylglucosamine desalkyl delavirdine.
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Fig. 6.
Contour plot of the 2D NMR COSY spectrum
(500 Mhz, methanol-d4) of the sugar region
of MET-4c.
Resonances for the unhydrolyzed MET-4c (bold) and free sugar
(normal) and MET-5 (normal) resulting from hydrolysis of MET-4c are
included.
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|
MET-4d showed a UV max at 302 nm and suggested the
presence of an indole ring. Sulfatase hydrolysis indicated that MET-4d contained a phenolsulfate group. The APCI and ESI mass spectra of
MET-4d (fig. 4, table 2) confirmed this assignment with a pseudomolecular ion at m/z 511, 96 amu higher than desalkyl
delavirdine and indicative of addition of a sulfate group. Loss of
sulfate gave an ion at m/z 431, subsequent cleavage of the
carbonyl-indole bond gave an ion at m/z 221. Cleavage of the
carbonyl-piperazine linkage resulted in ions at m/z 275 and
237, while cleavage of the piperazine-pyridine linkage gave an ion at
m/z 323. The m/z 323 ion was common to both
desalkyl delavirdine and MET-4d, while the m/z 275 ion was
96 amu higher than the corresponding ion for desalkyl delavirdine and
indicated the presence of a sulfate group on the pyridine ring. HPLC
retention time comparison with a previously isolated human urinary
metabolite finalized the structure of the monkey MET-4d as the
4-sulfate conjugate of desalkyl delavirdine (26).
MET-15b and MET-15d were observed upon treatment of MET-4b and MET-4d
with sulfatase. These data indicated that MET-15b and MET-15d were
6-hydroxy and 4-hydroxy desalkyl delavirdine, respectively. Assignments were confirmed by APCI-MS (table 2), which showed pseudomolecular ions for MET-15b and MET-15d at m/z 431, 16 amu higher than desalkyl delavirdine. Fragments arising from loss of
CH3SO2 (m/z 353), cleavage of the
carbonyl-piperazine linkage (m/z 195) and cleavage of the
carbonyl-indole bond (m/z 221) were 16 amu higher than
corresponding ions for desalkyl delavirdine and established
hydroxylation on the pyridine ring.
MET-16 was observed as a significant metabolite in monkey bile.
Treatment of bile with -glucuronidase (containing glucuronidase and
sulfatase activities) resulted in a decrease in MET-16. Upon treatment
of monkey bile with sulfatase (containing no detectable -glucuronidase activity), MET-16 remained unchanged. These data suggested that MET-6 was a glucuronic acid conjugate. Analysis of
MET-16 by ESI-MS-MS showed a pseudomolecular ion at m/z 649, 192 amu higher than parent drug, indicative of addition of a hydroxyl group followed by conjugation with glucuronic acid (fig. 3, table 2).
Loss of the glucuronic acid moiety gave an ion at m/z 473. Subsequent loss of CH3SO2 gave an ion at
m/z 394. Cleavage of the carbonyl-piperazine bond and the
carbonyl-indole linkage gave ions at m/z 221 and 247, respectively, and indicated substitution on the indole ring. To
determine the position of substitution, MET-16 was isolated from monkey
bile.
The purified MET-16 showed a UV max at 296 nm,
indicative of the presence of an indole ring. The 1H NMR
(500 MHz, methanol-d4, fig. 5, table 3) spectrum showed the
presence of six aromatic protons, eight piperazine protons, the
isopropyl side chain, a methylsulfonate group, and a sugar moiety. One
aromatic resonance was observed as a doublet of doublets with coupling
constants J6-4 and
J6-5 of 2.4 and 4.0 Hz, respectively, and was therefore assigned to the pyridine H-6
proton. A 2D COSY NMR experiment (table 4) showed homonuclear spin
coupling between H-6 and the multiplet at 6.98 ppm, integrating for
two protons, and assigned the resonance at 6.98 ppm to the pyridine
H-4 and H-5 protons. The remaining three aromatic resonances
corresponded to protons on the indole ring. The resonance at 7.35 ppm
appeared as a doublet with J3-7 of 0.8 Hz and was assigned
to the indole H-3 proton. The resonance at 7.25 ppm was observed as
a doublet of doublets with coupling constants J7-6 and
J3-7 of 8.8 and 0.8 Hz, respectively, and was assigned to
the indole H-7 proton. The remaining aromatic resonance at 7.32 ppm
corresponded to the indole H-6 proton and appeared as a doublet with
J6-7 of 8.7 Hz. Assignments were confirmed with 2D COSY
and 2D TOCSY NMR experiments. These spectroscopic data established that
MET-16 was substituted at the indole C-4. A 2D ROESY NMR experiment
confirmed this assignment, showing cross peaks between the indole H-3
proton and the anomeric sugar proton, and established that these
protons were in close steric proximity. The strong steric interaction
between the indole H-3 proton and the O-glucuronic acid
predicts that the electron cloud around H-3 becomes deformed resulting
in a shift to lower field. The 2D ROESY NMR experiment also indicated
that the sugar moiety was directed towards the pyridine ring and
accounted for the upfield shifts observed for the pyridine ring protons
relative to the parent drug. Additional resonances in the
1H NMR spectrum of MET-16 corresponded to the piperazine
ring protons ( 4.08 and 3.09 ppm), a methine proton ( 3.65), a
methyl group ( 1.26 ppm), a methylsulfonate group ( 2.93 ppm),
and a sugar moiety ( 4.86, 3.62, 3.59, 3.54, and 3.47). The methyl
protons in the isopropyl chain centered at 1.26 ppm were observed
as an apparent doublet of doublets with coupling constants of 1.7 and
6.3 Hz. However, further 1H NMR experiments at 300 and 400 MHz revealed that what appeared to be a doublet of doublets was
actually two doublets separated by 1.7 Hz (0.003 ppm) whose chemical
shifts migrated with frequency. These data indicated that the methyl
protons in the isopropyl chain were nonequivalent, probably because of
the steric proximity of the sugar. A coupling constant between the
anomeric H-1" ( 4.86 ppm) and H-2" ( 3.62 ppm) of 7.9 Hz
confirmed the -linkage of the sugar. Sugar resonances were assigned
with a 2D COSY NMR experiment (table 4). In addition, the coupling
constants J2"-3", J3"-4", and
J4"-5" of 8.8, 8.8 and 9.5 Hz, respectively, established axial conformations for H-2", H-3", H-4", and H-5" and identified MET-16 as 4--O-glucuronide delavirdine.
MET-17 was present as a minor metabolite in bile of monkeys treated
with delavirdine mesylate. Analysis of bile by LC-ESI-MS showed a
pseudomolecular ion for MET-17 at m/z 633, 176 amu higher than delavirdine and indicated addition of an N-linked
glucuronic acid. The position of glucuronidation was ascertained by
ESI-MS-MS (fig. 7). Loss of
CH3SO2 gave an ion at m/z 553, while
loss of glucuronic acid resulted in an ion at m/z 457. Cleavage of the piperazine-pyridine bond resulted in an ion at
m/z 499, 176 amu higher than the corresponding fragment in
the parent drug, indicating substitution of the piperazine, indole, or
sulfonamide nitrogens. Cleavage of the carbonyl-piperazine and
carbonyl-indole linkages gave ions at m/z 221 and 247, which
were also observed in the parent drug, and indicated substitution of
the indole or sulfonamide nitrogens. The ion at m/z 539 corresponded to loss of CH3SO2NH, and together
with the ions at m/z 221 and 247, established glucuronidation of the
indole N-1 nitrogen.
| |
Discussion |
Desalkyl delavirdine was the major drug-related material in
circulation after single-dose administration and in male monkeys after multiple-dose administration, with parent drug accounting for
most of the remaining radioactivity and conjugates of desalkyl delavirdine (MET-4) and 6-O-glucuronide delavirdine (MET-6)
as minor components.
Delavirdine was metabolized extensively by the monkey. Unchanged
delavirdine was not detected in urine. Desalkyl delavirdine accounted
for nearly half of the radioactivity in urine, with despyridinyl
delavirdine (MET-2) and conjugates of desalkyl delavirdine (MET-4)
accounting for most of the remaining radioactivity. In addition, the
indole carboxylic acid metabolite (MET-10), MET-3, and
6-O-glucuronide delavirdine (MET-6) were detected as minor components in monkey urine. Bile was mostly composed of desalkyl delavirdine and 6-O-glucuronide delavirdine, with parent
drug, 4-O-glucuronide delavirdine (MET-16), and conjugates
of desalkyl delavirdine (MET-4) as significant components. In addition,
several minor metabolites were observed in bile of delavirdine-treated monkeys.
The use of 1H NMR spectroscopy proved to be indispensable
for the identification of metabolites, especially to ascertain the location of glucuronidation and sulfation. Comparison of the chemical shifts and the splitting of the aromatic signals between delavirdine and the isolated metabolites (see fig. 5) was extremely useful in
assigning the structures of the metabolites, in view of the small
amounts isolated, the instability of some of the metabolites, and in
the absence of synthetic standards. Likewise, the use of tandem mass
spectrometry facilitated metabolite identification, establishing
substitution on either the pyridine or indole rings by comparison of
the fragmentation pattern between delavirdine and its metabolites.
Figure 8 illustrates the scheme for the metabolism of
delavirdine in the monkey. The metabolism of delavirdine in the monkey involves six pathways. First, N-desalkylation to desalkyl
delavirdine (MET-5, U-96183), followed by conjugation with sulfate
(MET-4a) or N-acetylglucosamine (MET-4c). Second,
hydroxylation of desalkyl delavirdine at the pyridine ring C-4
(MET-15d) or C-6 (MET-15b) and subsequent conjugation with sulfate
give MET-4d and MET-4b, respectively. Alternatively, hydroxylation of
delavirdine at the pyridine ring C-6 position to 6-hydroxy
delavirdine (MET-7) and subsequent conjugation with glucuronic acid
give 6-O-glucuronide delavirdine (MET-6). Third, the
pyridine ring in 6-hydroxy delavirdine is cleaved to give despyridinyl
delavirdine (MET-2, U-102466). Fourth, hydroxylation of delavirdine at
the indole ring C-4 position and conjugation with glucuronic acid to
4-O-glucuronide delavirdine (MET-16). Fifth,
N-glucuronidation of delavirdine at the indole ring
(MET-17). A sixth pathway, involves amide bond cleavage with release of
the N-isopropylpyridinepiperazine (MET-12, U-88703) and the
indole carboxylic acid (MET-10, U-96364) metabolites.
Of particular interest are the formation of the N-linked
-N-acetylglucosamine conjugate of desalkyl delavirdine,
the 4-O-glucuronide conjugate of delavirdine, and
N-glucuronidation of the indole nitrogen.
N-acetylglucosaminidation has been reported as a selective conjugation reaction for 7-hydroxylated bile acids such as
ursodeoxycholic acid (27-29), in humans. However, in the case of bile
acids, conjugation with N-acetylglucosamine occurs via
O-linkage. Hydroxylation of indoles has been reported at the 3-, 5-, 6-, and 7-positions (30-33). Indole 4-hydroxylation of
1-methyltetrahydro--carboline has been observed in vitro,
but not in vivo (34). The formation of this metabolite was
attributed to pretreatment of the rats from which hepatocytes were
isolated with 3-methylcholanthrene (34), a potent inducer of certain
forms of cytochrome P450. Likewise, the only precedence for
glucuronidation of the indole nitrogen is found for the carbazole
N-linked glucuronide identified in bile samples of rats
treated with carvedilol (35). Identification of the N-linked
-N-acetylglucosamine and 4-O-glucuronide
metabolites of delavirdine represents novel biotransformation pathways
for xenobiotics.
The authors gratefully acknowledge M. J. Prough, B. W. Jones, and K. E. Rousch for technical assistance with
animal dosimetry and sample collection. We also thank R. K. Jensen for
providing the monkey bile samples, R. S. P. Hsi, E. H. Chew, and J. A. Easter for synthesis of the 14C labeled drug, and L. J. Larion for preparation of the vehicle in the dosing formulations.
Abbreviations used are:
HIV-1, human
immunodeficiency virus type 1;
SPE, solid phase extraction;
PB, particle beam;
LC, liquid chromatography;
MS, mass spectrometry;
CI, chemical ionization;
ESI, electrospray ionization;
APCI, atmospheric
pressure chemical ionization;
2D, two-dimensional;
COSY, correlation
spectroscopy;
ROESY, rotating frame nuclear overhauser and exchange
spectroscopy;
TOCSY, total correlation spectroscopy;
LSC, liquid
scintillation counting.
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Abstract
Introduction
Abstract
