Steady-State Disposition of the Nonpeptidic Protease Inhibitor Tipranavir when Coadministered with Ritonavir

Abstract

The pharmacokinetic and metabolite profiles of the antiretroviral agent tipranavir (TPV), administered with ritonavir (RTV), in nine healthy male volunteers were characterized. Subjects received 500-mg TPV capsules with 200-mg RTV capsules twice daily for 6 days. They then received a single oral dose of 551 mg of TPV containing 90 microCi of [(14)C]TPV with 200 mg of RTV on day 7, followed by twice-daily doses of unlabeled 500-mg TPV with 200 mg of RTV for up to 20 days. Blood, urine, and feces were collected for mass balance and metabolite profiling. Metabolite profiling and identification was performed using a flow scintillation analyzer in conjunction with liquid chromatography-tandem mass spectrometry. The median recovery of radioactivity was 87.1%, with 82.3% of the total recovered radioactivity excreted in the feces and less than 5% recovered from urine. Most radioactivity was excreted within 24 to 96 h after the dose of [(14)C]TPV. Radioactivity in blood was associated primarily with plasma rather than red blood cells. Unchanged TPV accounted for 98.4 to 99.7% of plasma radioactivity. Similarly, the most common form of radioactivity excreted in feces was unchanged TPV, accounting for a mean of 79.9% of fecal radioactivity. The most abundant metabolite in feces was a hydroxyl metabolite, H-1, which accounted for 4.9% of fecal radioactivity. TPV glucuronide metabolite H-3 was the most abundant of the drug-related components in urine, corresponding to 11% of urine radioactivity. In conclusion, after the coadministration of TPV and RTV, unchanged TPV represented the primary form of circulating and excreted TPV and the primary extraction route was via the feces.

Full text

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 2007, p. 2436–2444 Vol. 51, No. 7
0066-4804/07/$08.00⫹0 doi:10.1128/AAC.01115-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Steady-State Disposition of the Nonpeptidic Protease Inhibitor
Tipranavir when Coadministered with Ritonavir
䌤
Linzhi Chen,
1
* John P. Sabo,
1
Elsy Philip,
1
Yanping Mao,
1
Stephen H. Norris,
1
Thomas R. MacGregor,
1
Jan M. Wruck,
3
Sandra Garfinkel,
2
Mark Castles,
1
Amy Brinkman,
4
and Hernan Valdez
2
Departments of Drug Metabolism and Pharmacokinetics,
1
Virology,
2
and Biometrics and Data Management,
3
Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut, and
Covance Laboratories Inc., Madison, Winsconsin
4
Received 4 September 2006/Returned for modification 28 January 2007/Accepted 30 April 2007
The pharmacokinetic and metabolite profiles of the antiretroviral agent tipranavir (TPV), administered with
ritonavir (RTV), in nine healthy male volunteers were characterized. Subjects received 500-mg TPV capsules
with 200-mg RTV capsules twice daily for 6 days. They then received a single oral dose of 551 mg of TPV
containing 90 ␮Ci of [
14
C]TPV with 200 mg of RTV on day 7, followed by twice-daily doses of unlabeled 500-mg
TPV with 200 mg of RTV for up to 20 days. Blood, urine, and feces were collected for mass balance and
metabolite profiling. Metabolite profiling and identification was performed using a flow scintillation analyzer
in conjunction with liquid chromatography-tandem mass spectrometry. The median recovery of radioactivity
was 87.1%, with 82.3% of the total recovered radioactivity excreted in the feces and less than 5% recovered from
urine. Most radioactivity was excreted within 24 to 96 h after the dose of [
14
C]TPV. Radioactivity in blood was
associated primarily with plasma rather than red blood cells. Unchanged TPV accounted for 98.4 to 99.7% of
plasma radioactivity. Similarly, the most common form of radioactivity excreted in feces was unchanged TPV,
accounting for a mean of 79.9% of fecal radioactivity. The most abundant metabolite in feces was a hydroxyl
metabolite, H-1, which accounted for 4.9% of fecal radioactivity. TPV glucuronide metabolite H-3 was the most
abundant of the drug-related components in urine, corresponding to 11% of urine radioactivity. In conclusion,
after the coadministration of TPV and RTV, unchanged TPV represented the primary form of circulating and
excreted TPV and the primary extraction route was via the feces.
Tipranavir (TPV) is a nonpeptidic (15, 16) sulfonamide-
substituted dihydropyrone (Fig. 1) protease inhibitor (PI) mar-
keted for the treatment of human immunodeficiency virus
(HIV)-infected, treatment-experienced patients under the
trade name Aptivus. Most viruses resistant to PIs retain sus-
ceptibility to TPV (1, 6, 12). Like other PIs, TPV binds directly
to HIV aspartyl protease, thereby disrupting the catalytic site
of the enzyme and preventing the protease-dependent cleav-
age of HIV gag and gag-pol polyproteins into smaller func-
tional proteins (10, 15, 16). Clinical studies have demonstrated
the significant activity of TPV against HIV type 1 in infected
patients receiving twice-daily doses ranging from 300 to 1,200
mg (2, 3, 4, 9).
In clinical studies with healthy volunteers and HIV-infected
patients, TPV pharmacokinetic parameters, including the
steady-state trough concentration, the area under the concen-
tration-time curve (AUC) for plasma, the maximum concen-
tration in plasma during the steady state (C
max
), and the ap-
parent terminal half-life (t
1/2
), were substantially improved
with the concomitant administration of ritonavir (RTV) (8, 9).
Since RTV strongly inhibits cytochrome P450 3A4 (CYP3A4)
(5), the boosted levels of TPV in plasma with the coadminis-
tration of TPV and RTV (TPV/r) indicate that the metabolism
of TPV occurs via the cytochrome P450 (CYP450) pathway (8,
9). Phase II studies have also demonstrated that TPV is an
inducer of CYP450 metabolism or intestinal P glycoprotein
efflux, thereby lowering systemic RTV concentrations when
administered with RTV (8, 11, 14). Clinical studies with
healthy volunteers and HIV-infected patients have evaluated
potential drug interactions of TPV/r and established that no
TPV dose adjustments are required when TPV/r is adminis-
tered in conjunction with other commonly employed antiret-
roviral agents (13; F. D. Goebel, J. P. Sabo, T. R. MacGregor,
D. L. Mayers, and S. McCallister, presented at the HIV DART
Conference, 2002).
The purpose of the present study was to characterize the
pharmacokinetic and metabolite profiles of TPV when admin-
istered with RTV.
MATERIALS AND METHODS
Materials. TPV, [
14
C]TPV, and TPV glucuronide were synthesized at Boehr-
inger Ingelheim Pharmaceuticals, Inc. (Ridgefield, CT). The position of the
14
C
label is indicated in Fig. 1. The chemical identities of the compounds were
established by high-performance liquid chromatography, mass spectrometry, and
nuclear magnetic resonance. The radiochemical purity of [
14
C]TPV was deter-
mined by liquid chromatography (LC)-radiochromatography to be ⬎98%. Aqua-
sol-2, Ultima Gold XR, and Ultima FLO-M scintillation cocktails were pur-
chased from PerkinElmer Life and Analytical Sciences (Shelton, CT). All other
chemicals were provided by standard commercial sources and were of reagent
grade or better.
Subjects, dosing, and sample collection. This phase I, open-label, multiple-
oral-dose study was conducted to characterize the pharmacokinetics of TPV and
its metabolites, to elucidate the disposition of the parent compound and the total
radioactivity by excretion and mass balance during the steady state, and to
* Corresponding author. Mailing address: Research and Develop-
ment, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury
Rd., Ridgefield, CT 06877. Phone: (203) 778-7870. Fax: (203) 791-
6003. E-mail: lchen@rdg.boehringer-ingelheim.com.
䌤
Published ahead of print on 7 May 2007.
2436

identify and quantify major metabolites of TPV in plasma, urine, and feces. The
study was conducted in accordance with the Nuclear Regulatory Commission
regulations and the Declaration of Helsinki (1964 and subsequent revisions). Of
the 12 healthy male volunteers enrolled, 9 completed all phases of the study,
including the administration of [
14
C]TPV/r. Subjects were nonsmokers and re-
ceived standardized meals and the same supervised doses of TPV/r, i.e., 500 mg
of TPV plus 200 mg of RTV twice daily for 6 days. On day 7, participants
received a light snack at 6:00 am, 60 min prior to the administration of 500 mg
of [
14
C]TPV (90 ␮Ci) plus 200 mg of RTV. Subjects fasted for an additional 4 h
after the dose administration. At 7:00 pm that evening, participants resumed
dosing with nonradioactive TPV at 500 mg and RTV at 200 mg twice daily and
continued on this dosing regimen up to day 20 of the study. Subjects did not
receive any medications known to affect CYP450 activity, and coffee, tea, cola,
chocolate, St. John’s wort, milk thistle, garlic supplements, Seville oranges,
grapefruit, grapefruit juice, and alcohol were avoided during the course of the
study.
Blood samples were collected daily, immediately prior to the evening doses of
TPV/r on study days 1 through 7 (5 ml) and prior to the morning doses on study
days 8 through 15 (10 ml). On day 7, serial 10-ml blood samples were obtained
10 min prior to the radioactive dose and at 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, and
24 h post-radioactive dose. Both whole blood and plasma were analyzed for
radioactivity. Additional 10-ml blood samples for metabolite profiling were col-
lected at 3, 8, and 12 h post-radioactive dose. The 3-h time point coincides with
the mean peak plasma TPV concentration (8), while the 8- and 12-h time points
were chosen for metabolite profiling as late time points near the end of the 12-h
dosing interval. Plasma was prepared immediately after sampling by centrifuga-
tion at 5°C for 10 min at 2,000 ⫻g.
After the administration of the radioactive dose, urine and feces were col-
lected until the end of the study. All samples were stored at approximately ⫺20°C
until analyzed.
Radioactivity measurement. Feces and tissue samples were homogenized in
water and a mixture of ethanol and water (1:1), respectively, which were approx-
imately three to five times the sample weight. Whole-blood, fecal homogenate,
and tissue homogenate samples were combusted before radioactivity counting
with a Packard 307 sample oxidizer (Packard Instrument Co., Meriden, CT), and
the resulting
14
CO
2
was trapped in a mixture of Perma Fluor and Carbo-Sorb
(Packard Instrument Co., Meriden, CT). Aliquots of samples (0.2 ml of blood
and plasma, 0.5 ml of urine, and 0.5 g of fecal and tissue homogenates) were
mixed with 5 ml of Ultima Gold XR scintillation cocktail and evaluated for
radioactivity by using Packard 2900TR liquid scintillation counters (Packard
Instrument Co.). Scintillation counting data (expressed in counts per minute)
were automatically corrected for counting efficiency by using the external stan-
dardization technique and an instrument-stored quench curve generated from a
series of sealed quenched standards.
Quantitation of TPV in plasma. The concentrations of TPV in plasma were
determined using a validated LC-tandem mass spectrometry (MS-MS) method.
Plasma samples were prepared by a two-step liquid-liquid extraction procedure;
the use of an ethyl acetate-hexane mixture was followed by a hexane wash.
Chromatographic separation was achieved using a 2.0- by 30-mm, 4-␮m-particle-
size Synergi Polar-RP column (Phenomenex Inc., Torrance, CA). The mobile
phase consisted of 50% 20 mM formic acid–10 mM acetic acid and 50% aceto-
nitrile; the run time was 6 min. The detector used was a MicroMass QuattroLC
triple-quadrupole mass spectrometer (Waters, Milford, MA), which was
equipped with an electrospray ion source and operated in positive-selective-
reaction monitoring mode. Key MS-MS operating parameters included the fol-
lowing: capillary voltage, 1 kV; nebulizer gas, 110 liters/h; desolvation gas, 1,300
liters/h; desolvation temperature, 300°C; cone voltage, 30 V; and collision offset,
20 V. Of the two overlapping calibration curves used in this study, one had a
calibration range of 25.0 to 2,000 ng/ml and the other had a calibration range of
1,000 to 20,000 ng/ml. Two calibration curves with different calibration ranges
were used to minimize sample manipulation during bioanalysis. All of the sam-
ples were assayed with the higher-range curve first. For the samples that had a
concentration of less than 1,000 ng/ml (the lower limit of quantitation of the
higher-range curve), there was no reportable result, and these samples were
subsequently reassayed with the lower-range curve.
Metabolite sample extraction. The plasma samples collected from the nine
subjects at each of the three time points, 3, 8, and 12 h postdose, were pooled for
metabolite profiling and identification. A 20- to 25-ml aliquot of the plasma pool
was evaporated at room temperature under a nitrogen stream in a Zymark
TurboVap LV evaporator (Zymark, Hopkinton, MA), and the residue was then
extracted four to six times with 10 to 20 ml of methanol. For each extraction, the
mixture was sonicated for 5 min, and the supernatant was separated by centrif-
ugation. The extracts were combined, evaporated with the TurboVap, and sub-
jected to further cleanup via multiple evaporation-extraction cycles with meth-
anol and acetonitrile. The final sample was reconstituted in 0.30 to 0.43 ml of
methanol and transferred to an autosampler vial for LC-radiochromatography-
MS-MS analysis. The total extraction recovery of radioactivity from plasma
ranged from 87.8 to 100.8%.
The urine samples collected from each subject during the first 24 h after
[
14
C]TPV dosing were pooled. The majority (50.0 to 63.3%) of urine radioac-
tivity was found in the urine from the 0-to-24-h sample collection period, and the
remaining radioactivity was spread throughout large volumes of urine across
several days. Equal percentages (by volume) of the 0-to-24-h urine samples were
combined to make the pools used for metabolite profiling and identification. To
a 100-ml aliquot of pooled urine was added 6 ml of 28 to 30% ammonium
hydroxide. The urine samples were then extracted via solid-phase extraction with
35-cm
3
/6-g Oasis HLB solid-phase extraction cartridges (Waters Corporation,
Millford, MA). After loading, the solid-phase extraction cartridge was washed
with 25 to 35 ml of 2% ammonium hydroxide, and the sample was eluted with
methanol. The eluate was evaporated and further cleaned up via multiple evap-
oration-extraction cycles with acetonitrile. The final samples were reconstituted
in 0.4 to 0.5 ml of methanol and transferred to an autosampler vial for metabolite
profiling and identification. The total extraction recovery of radioactivity from
urine ranged from 84.1 to 101.7%.
A fecal pool for each subject for metabolite profiling and identification was
also prepared by combining aliquots of fecal homogenates from three to five
sampling time points at equal percentages (by volume). Depending on the sub-
ject, feces from 0 to 24 h up to 120 to 144 h were pooled. The pools corresponded
to 90% or more of the total radioactivity ultimately excreted in feces. An 18- to
30-ml aliquot of the pooled fecal homogenate was centrifuged, the supernatant
was discarded, and the pellet was extracted with acetonitrile, methanol, and
acidified methanol. The extracts were combined, evaporated, and further cleaned
up via multiple evaporation-extraction cycles with acetonitrile. The final samples
were reconstituted in 0.3 to 0.5 ml of methanol and then transferred to an
autosampler vial for metabolite profiling and identification. The total extraction
recovery of radioactivity from feces ranged from 81.2 to 106.1%.
Metabolite profiling and identification. Metabolite profiling and identification
was carried out with an LC-radiochromatography-MS-MS system, which con-
sisted of an Agilent 1100 series high-performance liquid chromatography system
(Agilent, Palo Alto, CA) with a Packard 525 Radiomatic flow scintillation ana-
lyzer (Packard Instrument Co.) and a Finnigan LCQ Deca XP
⫹
ion trap mass
spectrometer (Finnigan, San Jose, CA) as detectors. The chromatographic sep-
aration was achieved with an xTerra MS C
18
column, 250 by 4.6 mm, with a 5-␮m
particle size (Waters Corporation). The mobile phase A comprised 0.1% acetic
acid containing 5% acetonitrile, and the mobile phase B was acetonitrile con-
taining 0.1% acetic acid. The gradient conditions were 18 to 50% mobile phase
B over 50 min and then to 100% over 20 min at 1 ml/min. The post column flow
was split in a 1:20 ratio via an Acurate flow splitter (LC Packings, San Francisco,
CA); 20 parts were sent to the flow scintillation analyzer and 1 part was sent to
the ion trap mass spectrometer. The flow scintillation analyzer was equipped with
a 250-␮l flow cell, and the detection window was 4 to 100 keV. Packard Ultima
FLO-M was used as the scintillation cocktail, and the flow rate was 2.5 ml/min.
The LCQ mass spectrometer was equipped with an electrospray ion source and
operated in positive mode. Key operating parameters included a spray voltage of
5 kV, a sheath gas flow of 35 U, an auxiliary gas flow of 0 U, a capillary
temperature of 300°C, and an MS-MS collision energy of 40% with MS-MS wide
excitation.
Pharmacokinetic analysis. The pharmacokinetic profiles of TPV were deter-
mined using plasma and blood TPV concentrations in a noncompartmental
model (WinNonlin; Pharsight Corporation, Mountain View, CA). The following
pharmacokinetic parameters for TPV in plasma and blood were analyzed: morn-
ing and evening trough concentrations, the C
max
, the concentration in plasma
12 h after dosing (C
12h
), the AUC, the time to maximum concentration (T
max
),
and the t
1/2
of total
14
C radioactivity.
FIG. 1. Structure of the sulfonamide-substituted dihydropyrone PI
TPV. The asterisk denotes the
14
C label.
VOL. 51, 2007 TIPRANAVIR-RITONAVIR DISPOSITION 2437

RESULTS
Mass balance and excretion of radiolabel. A total of nine
male subjects (eight white, one black) from 22 to 53 years of
age with body mass indices of 21 to 27 kg/m
2
received radio-
labeled TPV. Approximately 75.3% ⫾23.7% (mean ⫾stan-
dard deviation [SD]) of the radiolabeled dose was recovered
from feces, urine, and tissue (Table 1; Fig. 2, inset). Subject 113
was excluded from the mass balance analyses because diarrhea
prevented complete sample collection. Subject 109 had 21.3%
of the radiolabeled dose recovered, and this result was an
outlier for the study group as a whole. For the remaining seven
subjects, the total recovery of radioactivity was 83.0% ⫾
10.0%. Feces represented the primary route of excretion, with
70.4% ⫾24.0% of the total radioactivity dose recovered via
this route. Urinary excretion accounted for 4.6% ⫾0.6% of the
total administered radioactive dose.
Radioactivity and TPV concentrations in plasma and blood.
The pharmacokinetic parameters for TPV and for total radio-
activity in plasma and blood are shown in Tables 2 and 3. Plots
of mean concentrations of radioactivity in plasma and whole
blood versus time are shown in Fig. 2. For all subjects, con-
centrations of radioactivity in plasma and blood declined
steadily to below the limit of quantitation by 96 and 120 h
postdose, respectively. The plots of blood and plasma radioac-
tivity concentrations versus time were parallel, and the ratio of
the concentration of circulating radioactivity in plasma to that
in blood was 1.89 ⫾0.08 at each time point. Therefore, ap-
proximately 85% of the TPV in blood was distributed in the
plasma, with the remaining TPV in erythrocytes, and the dis-
tribution between plasma and blood cells was approximately
constant. The plasma radioactivity-time profile could also be
superposed on the plasma TPV concentrations obtained using
FIG. 2. Radioactivity in plasma (E) and blood (F) after a single oral dose of [
14
C]TPV administered during the steady state. Means ⫾SDs
are shown (n⫽9). (Inset) Cumulative excretion of radioactivity in urine (䡺) and feces (E) and total radioactivity (F). Means ⫾SDs are shown
(n⫽8).
TABLE 1. Summary of cumulative recovery of radioactivity from samples from human subjects after oral administration of [
14
C]TPV
Sample type Cumulative % of radioactive dose recovered from subject: Mean
a
SD
104 106 108 109 113
a
116 122 123 127
Urine 5.07 4.94 4.39 5.60 3.88 4.21 4.36 4.50 3.64 4.59 0.60
Feces 84.4 62.2 81.4 15.7 32.3 87.0 65.3 83.2 83.8 70.4 24.0
Toilet tissue specimen 0.79 0.30 0.71 0.01 0.07 0.04 0.07 0.77 0.23 0.37 0.34
Total 90.3 67.4 86.5 21.3 36.3 91.3 69.7 88.5 87.7 75.3 23.7
a
Subject 113 had an episode of explosive diarrhea on day 13, and the sample was lost. Thus, the results for subject 113 were excluded from the calculationsofall
the means involving excreta.
2438 CHEN ET AL. ANTIMICROB.AGENTS CHEMOTHER.

the LC-MS-MS assay, indicating that the abundance of TPV
relative to the total level of radioactivity in plasma remained
approximately constant.
Pharmacokinetics. The pharmacokinetic profiles of TPV
were characterized by using the concentration data for
plasma as measured before and after the administration of
the multiple-dose regimen and the single 551-mg oral dose
of [
14
C]TPV. Pharmacokinetic data derived prior to the
administration of radiolabeled TPV suggested that the
steady-state plasma TPV concentration was achieved by day
7 of the twice-daily administration of TPV/r at 500 and 200
mg, respectively (Fig. 3). The range of trough plasma TPV
TABLE 2. Pharmacokinetic parameters for TPV in plasma
Parameter
a
Mean SD Minimum Median Maximum Geometric mean
d
Total TPV dose (mg) 551 1 549 551 552
T
max
(h) 2.9 1.1 1.5 3.0 5.0 2.8
C
max
(␮M) observed
b
99.39 23.37 74.93 92.08 141.39 97.09
C
max
(␮M) normalized
c
90.23 21.30 67.87 83.56 128.31 88.13
C
12h
(␮M) observed
b
25.85 18.84 6.94 22.49 72.27 21.44
C
12h
(␮M) normalized
c
23.46 17.06 6.29 20.41 65.46 19.46
AUC
0–12
(h 䡠␮M) observed
b
657.0 170.9 452.5 601.2 922.9 638.4
AUC
0–12
(h 䡠␮M) normalized
c
596.5 155.7 409.9 545.5 840.6 579.4
␭
z
(h
⫺1
)0.1631 0.0499 0.0952 0.1713 0.2495 0.1561
t
1/2
(h) 4.6 1.5 2.8 4.0 7.3
CL (liters/h) 1.47 0.36 0.99 1.52 2.02 1.43
V(liters) 9.6 3.2 4.9 9.2 16.3 9.2
a
Results from nine subjects were included in the calculations. The total TPV dose includes unlabeled TPV and [
14
C]TPV. ␭
z
, first-order rate constant associated
with the terminal (log-linear) elimination phase; CL, clearance; V, volume of distribution.
b
Based on noncompartmental pharmacokinetic analysis using reported TPV concentrations.
c
Based on noncompartmental pharmacokinetic results corrected for the TPV dose, as follows: (observed pharmacokinetic metric/actual TPV dose) 䡠nominal TPV
dose, where the actual TPV dose is the dose administered to each subject and the nominal TPV dose is 500 mg.
d
The harmonic mean t
1/2
was 4.2 h.
TABLE 3. Pharmacokinetic parameters for radioactivity in plasma and blood
a
Biomatrix and
time period Value T
max
(h)
T
last
(h)
C
max
(␮M eq)
C
last
(␮M eq)
C
12h
(␮M eq)
␭
z
(h
⫺1
)
t
1/2
(h)
AUC
(h 䡠␮M eq)
CL
(liters/h)
V
(liters)
MRT
(h)
Blood, 0–12 h Mean 2.61 49.45 10.26 0.1694 4.20 297.35 2.64 15.68
SD 0.60 11.19 3.34 0.0296 0.74 61.52 0.56 2.65
Min 1.50 35.92 4.96 0.1210 3.05 233.85 1.78 10.25
Median 3.00 47.13 9.58 0.1653 4.19 277.18 2.69 15.89
Max 3.00 74.99 15.33 0.2272 5.73 429.04 3.48 19.87
gMean 2.54 48.47 9.75 0.1672 4.15 292.27 2.59 15.46
hMean 4.09
Plasma, 0–12 h Mean 2.56 90.55 18.50 0.1761 4.07 546.97 1.45 8.32
SD 0.53 15.98 6.54 0.0374 0.74 111.16 0.30 1.57
Min 2.00 70.99 7.30 0.1348 2.63 439.66 0.96 5.81
Median 3.00 90.57 18.39 0.1752 3.96 531.54 1.44 8.12
Max 3.00 122.91 28.77 0.2640 5.14 779.57 1.88 10.75
gMean 2.51 89.37 17.33 0.1730 4.01 537.81 1.42 8.18
hMean 3.94
Blood, 0–⬁Mean 64.00 0.93 0.0482 16.16 492.45 1.98 44.23 16.56
SD 16.97 0.31 0.0236 4.27 127.75 0.56 12.75 4.15
Min 24.00 0.62 0.0358 6.36 288.85 1.29 29.09 7.18
Median 72.00 0.91 0.0399 17.37 449.10 2.03 42.79 16.96
Max 72.00 1.44 0.1090 19.38 709.82 3.17 63.41 22.64
gMean 60.92 0.89 0.0449 15.43 477.46 1.91 42.61 15.94
hMean 14.37
Plasma, 0–⬁Mean 90.67 0.82 0.0334 21.32 910.92 1.06 32.47 18.29
SD 10.58 0.20 0.0058 3.55 220.61 0.25 9.61 2.66
Min 72.00 0.60 0.0268 16.58 600.71 0.70 18.98 13.48
Median 96.00 0.74 0.0327 21.22 891.71 1.03 30.93 18.40
Max 96.00 1.13 0.0418 25.87 1310.61 1.52 51.67 23.63
gMean 90.05 0.80 0.0329 21.05 887.71 1.03 31.27 18.11
hMean 20.78
a
Results for nine subjects were included in the calculations. Min, minimum; max, maximum; gMean, geometric mean; hMean, harmonic mean; T
last
, time of last
measurable concentration; C
last
, last measurable concentration; ␭
z
, first-order rate constant associated with the terminal (log-linear) elimination phase; CL, clearance;
V, volume of distribution; MRT, mean residence time.
VOL. 51, 2007 TIPRANAVIR-RITONAVIR DISPOSITION 2439

concentrations for all subjects following the first two doses
of TPV/r was 30.6 to 98.4 ␮M. TPV trough concentrations
increased through three additional dose administrations
with continued twice-daily dosing of TPV/r to a geometric
mean of 70.0 ␮M on the evening of day 2, which was reflec-
tive of CYP3A inhibition by RTV. Mean evening trough
concentrations decreased by 6 ␮M per day for the next 4
days, and by day 7 the trough TPV concentration was 21.9
␮M. This level was indicative of the establishment of equi-
librium between CYP3A inhibition by RTV and induction
by TPV. A steady state was established by day 7, as the
geometric mean ratio of the day 7 evening measurement to
the day 8 morning measurement was 1.07. Geometric mean
morning trough TPV concentrations from day 8 through day
15 averaged 18.2 ␮M (range, 16.6 to 21.9 ␮M).
The calculated pharmacokinetic parameters for TPV are
shown in Table 2, and those for total radioactivity are pre-
sented in Table 3. Using noncompartmental analyses, the me-
dian T
max
was calculated as 3.0 h (range, 1.5 to 5.0 h), with a
median normalized C
max
of 83.56 ␮M (range, 67.87 to 128.31
␮M) and a median normalized C
12h
of 20.41 ␮M (range, 6.29
to 65.46 ␮M). The values were normalized for the actual ra-
dioactive dose (551 mg) given to the subjects as opposed to the
nominal 500-mg dose given on the other days. The median
elimination t
1/2
of TPV in plasma of 4.0 h (range, 2.8 to 7.3 h)
closely approximated the median elimination t
1/2
of radioac-
tivity in plasma of 3.96 h (range, 2.63 to 5.14 h) and that in
whole blood of 4.19 h (range, 3.05 to 5.73 h). For this group of
subjects, the median AUC from 0 to 12 h (AUC
0–12
) following
TPV/r administration at 500 and 200 mg was 545.5 h 䡠␮M
(range, 409.9 to 840.6 h 䡠␮M).
TPV metabolites. Unchanged TPV was the predominant
circulating component in the pooled plasma samples at 3, 8,
and 12 h postdose and accounted for 98.4 to 99.7% of the
plasma radioactivity (Table 4; Fig. 4a). Only a few trace me-
tabolites, including a TPV glucuronidation conjugate (H-3)
and an oxidation metabolite (H-1), were observed in radio-
chromatograms, and each metabolite accounted for ⱕ0.2%
of the plasma radioactivity. Based on this observation of
trace metabolites, it can be surmised that the plasma sam-
ples from all the subjects contained predominantly un-
changed drug and that pooling was necessary for metabolite
identification. Unchanged TPV also dominated the radio-
chromatograms for feces (Fig. 4c), accounting for 79.9% of
fecal radioactivity or, on average, 53.5% of the dose in the
nine subjects. Several metabolites were found in feces. Me-
tabolite H-1, the most abundant, was responsible for 3.2%
of the dose, while another oxidation metabolite, H-2, ob-
served in the feces from most of the subjects, represented
0.4% of the dose. The remaining metabolites in feces each
were less than 1.3% of the dose. In contrast, TPV was much
less abundant in urine and represented only 0.5% of urine
radioactivity or ⬍0.1% of the dose (Fig. 4b). The majority of
radioactivity in urine was accounted for by many metabo-
lites. TPV glucuronide conjugate (H-3), accounting for 0.5%
of the dose, was the most abundant urinary metabolite; the
remaining urinary metabolites were each less than 0.4% of
the dose.
The TPV glucuronide conjugate metabolite (H-3) corre-
sponded to a radiochromatographic peak at approximately
52.5 min and a protonated molecular ion at m/z 779, 176
daltons higher than that corresponding to TPV. Upon collision
activation, the molecular ion gave protonated TPV via the loss
of glucuronide. The identification of H-3 was confirmed by a
comparison of the retention time and the MS-MS pattern to
the authentic standard.
Metabolite H-1 had a protonated molecular ion at m/z 619,
16 daltons higher than that of TPV (Fig. 5). MS-MS analysis of
H-1 produced daughter ions at m/z 575 (loss of CO
2
) and 411
FIG. 3. Steady-state concentrations of TPV in plasma (Cp). Solid
line, geometric mean; dashed line, median; day 1 to day 6, evening
trough concentrations; day 7, morning and evening trough concentra-
tions (evening concentrations were not normalized to the TPV dose of
approximately 551 mg per subject); day 8 to day 15, morning trough
concentrations (n⫽7).
TABLE 4. Summary of abundance of radioactive metabolites in plasma, urine, and feces
Metabolite % of radioactivity (concn)
a
in plasma at: % of radioactivity
(% of radioactive dose) in excreta Total % of radioactive
dose in excreta
3 h 8 h 12 h Urine Feces
H-1 0.2 (0.10) ND ND ⬍0.1 (⬍0.1) 4.9 (3.2) 3.2
H-2 ND
b
ND ND ND 0.8 (0.4) 0.4
H-3 0.1 (0.05) ND ND 11.0 (0.5) ND 0.5
TPV 98.9 (48.6) 98.4 (21.0) 99.7 (10.7) 0.5 (⬍0.1) 79.9 (53.5) 53.5
Total 99.2 (48.8) 98.4 (21.0) 99.7 (10.7) 11.5 (0.5) 85.6 (57.1) 57.6
a
Concentrations of metabolites are expressed as microgram equivalents of [
14
C]TPV per milliliter.
b
ND, not detected.
2440 CHEN ET AL. ANTIMICROB.AGENTS CHEMOTHER.

via the same fragmentation patterns as TPV. In addition, new
fragments were observed at m/z 557 (loss of CO
2
and loss of
H
2
O) and 495 (loss of C
7
H
6
O and loss of H
2
O). The mass
spectral analysis suggests that H-1 was likely a hydroxyl me-
tabolite. Similarly, H-2 was tentatively identified as another
oxidation metabolite, as illustrated in Fig. 5.
Safety. Nausea and loose stools were the most common
adverse events associated with TPV/r administration. There
were no serious adverse events, and only two subjects prema-
turely discontinued the use of the study drug, one due to nausea
and alanine aminotransferase elevation and the other due to
alanine aminotransferase elevation. The most common labora-
tory abnormalities were increases in transaminases (greater than
or equal to Division of AIDS grade 3 in two subjects, or 17%) and
mild increases in cholesterol and triglycerides.
DISCUSSION
In this study conducted with healthy male volunteers dosed
with twice-daily TPV/r at 500 and 200 mg, steady-state con-
centrations in plasma were achieved by day 7. Upon initial
dosing, trough TPV concentrations were higher than those
during the steady state, which is consistent with immediate
CYP3A inhibition by RTV (5, 8). Trough TPV concentrations
steadily decreased with twice-daily dosing until day 7 and
thereafter remained relatively constant, reflecting steady-state
attainment, through the remainder of the study (day 15). The
progressive decrease in the trough TPV concentration and
subsequent plateau suggest either CYP3A induction by TPV
and RTV or a decreased bioavailability of TPV and RTV,
followed by a balance of the discordant effects of TPV and
RTV on metabolism and absorption. The trough TPV concen-
trations and other pharmacokinetic characteristics observed in
this trial are consistent with those characterized in other clin-
ical studies of TPV at similar TPV/r doses (8, 9).
After the administration of the [
14
C]TPV/r dose on day 7
(steady state), TPV was rapidly absorbed, and concentrations
of both TPV and the radioactivity in plasma peaked at around
3 h postdose. TPV was found to be the predominant circulating
component in plasma, accounting for more than 98.4% of
plasma radioactivity at 3, 8, and 12 h postdose.
FIG. 4. Representative radiochromatograms for the 3-h plasma sample pooled from all nine subjects (a) and the urine (b) and feces (c) samples
from subject 106 after the administration of the [
14
C]TPV dose.
VOL. 51, 2007 TIPRANAVIR-RITONAVIR DISPOSITION 2441

Approximately 75.3% of the radiolabeled dose was recov-
ered from feces, urine, and tissue combined, and most was
excreted by 96 h after dosing. In general, the level of recovery
was lower than expected, perhaps as a result of incomplete
sample collection by subjects. Fecal excretion, which accounted
for approximately 70.4% of the radioactive dose, was the pri-
mary elimination route for TPV. On the other hand, urinary
excretion was responsible for 4.6% of the radioactive dose and
represented only a minor route of elimination. The absolute
bioavailability of TPV, in the presence of RTV, is not known.
The predominant recovery from feces in this study may indi-
cate extensive biliary excretion or pronounced P glycoprotein
efflux during the steady state. Although several metabolites
existed in feces, each represented ⱕ3.2% of the dose. Addi-
tionally, all metabolites in urine were minor, with each ac-
counting for ⱕ0.5% of the dose. The proposed metabolic path-
ways of TPV, in the presence of RTV, are shown in Fig. 6. The
minor contribution of the kidneys to the elimination of TPV
suggests that adjustments of the TPV/r dosage would be un-
likely to be necessary for renally impaired patients.
The lack of significant TPV metabolism during the steady
state in the presence of RTV coadministration and the signif-
icant decline in trough TPV concentrations from the initial
dosing until the steady state was attained on day 7 suggest that
the fractions of TPV and RTV absorbed are lower during the
steady state than after a single dose. TPV (11) and, to a lesser
extent, RTV (7) are substrates for P glycoprotein efflux trans-
port. In a drug interaction study with the P glycoprotein sub-
strate loperamide, TPV exhibited P glycoprotein self-induction
capability (11). In long-term clinical trials with TPV and RTV,
TPV has been shown to significantly reduce systemic RTV
exposure (14), presumably due to P glycoprotein efflux trans-
port.
This study demonstrated that the vast majority of the ad-
ministered dose of TPV remains unchanged in plasma and
excrements. The lack of TPV metabolism after the adminis-
tration of TPV with RTV would be expected because of the
profound inhibition of CYP3A by RTV (5). Previous studies
have suggested that the first-pass metabolism of TPV, without
RTV coadministration, occurs via the CYP3A pathway (8, 9),
producing a maximum exposure of plasma to TPV of less than
1␮M. In addition, this study did not identify any major, un-
expected TPV metabolites from an alternative pathway that
might potentially have altered the pharmacokinetics, efficacy,
and safety of TPV.
The major findings of this study, the unchanged nature of
the majority of the administered dose in the plasma and the
lack of any major, unexpected TPV metabolites, indicate that
the structure of TPV remains intact and available to bind to
HIV protease. The favorable TPV pharmacokinetic and me-
FIG. 5. Comparison of MS-MS results for the TPV standard (a) and metabolites H-1 (b) and H-2 (c).
2442 CHEN ET AL. ANTIMICROB.AGENTS CHEMOTHER.

tabolite profiles demonstrated in this study, along with the
distinct structure and resistance profile of TPV, support TPV/r
as an important treatment option for HIV-infected patients
with resistance to multiple PIs.
REFERENCES
1. Back, N. K. T., A. van Wijk, D. Remmerswaal, M. van Monfort, M. Nijhuis,
R. Schuurman, and C. A. B. Boucher. 2000. In-vitro tipranavir susceptibility
of HIV-1 isolates with reduced susceptibility to other protease inhibitors.
AIDS 14:101–102.
2. Cahn, P., J. Villacian, A. Lazzarin, C. Katlama, B. Grinsztejn, K. Arasteh, P.
Lopez, N. Clumeck, J. Gerstoft, N. Stavrianeas, S. Moreno, F. Antunes, D.
Neubacher, and D. Mayers. 2006. Ritonavir-boosted tipranavir demonstrates
superior efficacy to ritonavir-boosted protease inhibitors in treatment-expe-
rienced HIV-infected patients: 24-week results of the RESIST-2 trial. Clin.
Infect. Dis. 43:1347–1356.
3. Gathe, J., D. A. Cooper, C. Farthing, D. Jayaweera, D. Norris, G. Pierone,
C. R. Steinhart, B. Trottier, S. L. Walmsley, C. Workman, G. Mukwaya, V.
Kohlbrenner, C. Dohnanyi, S. McCallister, and D. Mayers for the RESIST-1
Study Team. 2006. Efficacy of the protease-inhibitors tipranavir plus ritona-
vir in treatment-experienced patients: 24-week analysis from the RESIST-1
trial. Clin. Infect. Dis. 43:1337–1346.
4. Gathe, J. C., G. Pierone, P. Piliero, K. Arasteh, R. Rubio, R. G. LaLonde, D.
Cooper, A. Lazzarin, V. M. Kohlbrenner, C. Dohnanyi, J. Sabo, and D.
Mayers. 2007. Efficacy and safety of three doses of tipranavir boosted with
ritonavir in treatment-experienced HIV type-1-infected patients. AIDS Res.
Hum. Retrovir. 23:216–223.
5. Koudriakova, T., E. Iatsimirskaia, I. Utkin, E. Gangl, P. Vouros, E.
Storozhuk, D. Orza, J. Marinina, and N. Gerber. 1998. Metabolism of the
human immunodeficiency virus protease inhibitors indinavir and ritonavir by
human intestinal microsomes and expressed cytochrome P450 3A4/3A5:
mechanism-based inactivation of cytochrome P450 3A by ritonavir. Drug
Metab. Dispos. 26:552–561.
6. Larder, B. A., K. Hertogs, S. Bloor, C. van den Eynde, W. DeCian, Y. Wang,
W. W. Freimuth, and G. Tarpley. 2000. Tipranavir inhibits broadly protease
inhibitor-resistant HIV-1 clinical samples. AIDS 14:1943–1948.
7. Lee, C. G. L., M. M. Gottesman, C. O. Cardarelli, M. Ramachandra, K.-T.
Jeang, S. V. Ambudkar, I. Pastan, and S. Dey. 1998. HIV-1 protease inhib-
itors are substrates for the MDR1 multidrug transporter. Biochemistry 37:
3594–3601.
8. MacGregor, T. R., J. P. Sabo, S. H. Norris, P. Johnson, L. Galitz, and S.
McCallister. 2004. Pharmacokinetic characterization of different dose com-
binations of coadministered tipranavir and ritonavir in healthy volunteers.
HIV Clin. Trials 5:371–382.
9. McCallister, S., H. Valdez, K. Curry, T. MacGregor, M. Borin, W. Freimuth,
Y. Wang, and D. L. Mayers. 2004. 14-day dose-response study of the efficacy,
safety, and pharmacokinetics of the nonpeptidic protease inhibitor tipranavir
in treatment-naive HIV-1-infected patients. J. Acquir. Immune Defic. Syndr.
35:376–382.
10. Monini, P., C. Sgadari, G. Barillari, and B. Ensoli. 2003. HIV protease
inhibitors: antiretroviral agents with anti-inflammatory, anti-angiogenic and
anti-tumour activity. J. Antimicrob. Chemother. 51:207–211.
11. Mukwaya, G., T. R. MacGregor, D. Hoelscher, T. Heming, D. Legg, K.
Kavanaugh, P. Johnson, J. P. Sabo, and S. McCallister. 2005. Interaction of
ritonavir-boosted tipranavir with loperamide does not result in loperamide-
associated neurologic side effects in healthy volunteers. Antimicrob. Agents
Chemother. 49:4903–4910.
12. Poppe, S. M., D. E. Slade, K. T. Chong, R. R. Hinshaw, P. J. Pagano, M.
Markowitz, D. D. Ho, H. Mo, R. R. Gorman III, T. J. Dueweke, S. Thais-
rivongs, and W. G. Tarpley. 1997. Antiviral activity of the dihydropyrone
PNU-140690, a new nonpeptidic human immunodeficiency virus protease
inhibitor. Antimicrob. Agents Chemother. 41:1058–1063.
13. Roszko, P. J., K. Curry, B. Brazina, A. Cohen, E. L. Turkie, J. P. Sabo, T. R.
FIG. 6. Proposed metabolic pathways of TPV administered with RTV. Gluc, glucuronide.
VOL. 51, 2007 TIPRANAVIR-RITONAVIR DISPOSITION 2443

MacGregor, and S. McCallister. 2003. Standard doses of efavirenz (EFV),
zidovudine (ZDV), tenofovir (TDF), and didanosine (ddI) may be given with
tipranavir/ritonavir (TPV/r). Antivir. Ther. 8:S428.
14. Sabo, J. P., P. J. Piliero, A. Lawton, T. R. MacGregor, and J. Leith. 2006. A
comparison of steady-state trough plasma ritonavir concentrations for HIV⫹
patients receiving an optimized background regimen and ritonavir-boosted
tipranavir (TPV/r), lopinavir (LPV/r), saquinavir (SQV/r) or amprenavir
(APV/r), poster 43. In Proceedings of the 7th International Workshop on
Clinical Pharmacology of HIV Therapy (Lisbon). Virology Education, Utre-
cht, The Netherlands.
15. Thaisrivongs, S., and J. W. Strohbach. 1999. Structure-based discovery of
tipranavir disodium (PNU-140690E): a potent, orally bioavailable, nonpep-
tidic HIV protease inhibitor. Biopolymers 51:51–58.
16. Turner, S. R., J. W. Strohbach, R. A. Tommasi, P. A. Aristoff, P. D.
Johnson, H. I. Skulnick, L. A. Dolak, E. P. Seost, P. K. Tomich, M. J.
Bohanon, M. M. Horng, J. C. Lynn, K. T. Chong, R. R. Hinshaw, K. D.
Watenpaugh, M. N. Janakiraman, and S. Thaisrivongs. 1998. Tipranavir
(PNU-140690): a potent, orally bioavailable nonpeptidic HIV protease
inhibitor of the 5,6-dihydro-4-hydroxy-2-pyrone sulfonamide class. J. Med.
Chem. 41:3467–3476.
2444 CHEN ET AL. ANTIMICROB.AGENTS CHEMOTHER.