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Introduction
Human immunodeficiency virus (HIV) infection is now a
major public health problem for both developing and
deve-loped countries as it has dramatically increased the global
burden of disease[1-5]. Many efforts have been made on
research and discovery of anti-HIV drugs; however, it still
remains difficult to develop more effective, affordable, and
less toxic anti-HIV drugs for the increasing number of
HIV-infected patients[6].
In recent years, more and more researchers have fixed
their eyes on preventing the entry of HIV into a host cell at
the step of gp41-mediated viral and cellular membrane fusion,
which was denoted as fusion
inhibitors[7]. The Food and Drug Administration of the United States announced, in 2003,
the accelerated approval of enfuvirtide, the first fusion
inhibitor, for use in combination with other anti-HIV
medications to treat advanced HIV-1 infection in adults and
children aged 6 years and older[8]. Enfuvirtide is administered
twice daily by subcutaneous injection into the abdomen,
the upper arm, or the anterior thigh for clinical
therapeutics[9].
Sifuvirtide is a linear 36-amino acid peptide with an amino
acid sequence of Acetyl-SWETWEREIENYTRQIYRILEE-SQEQQDRNERDLLE. It is a novel antiviral peptide that has
been classified as a fusion inhibitor. The profiles of
absorption, distribution, metabolism and excretion of
sifuvirtide have been characterized in rats and monkeys by
using radioactive 125I-sifuvirtide combined with
radioactivity detection of samples derived from trichloroacetic acid
precipitation or size-exclusion chromatography. For the
requirement of clinical pharmacokinetic (PK) study, we have
developed an advanced and validated on-line solid-phase
extraction procedure combined with liquid chromatography
tandem mass spectrometry (SPE-LC/MS/MS) approach for
determination of non-labeled sifuvirtide in monkey
plasma[10].
In the present study, an in vitro fusion inhibitory
experiment was used to determine the 50% inhibitory
concentration (IC50) of sifuvirtide. According to the results of 50 times
lower IC50 value than enfuvirtide and the pharmacological
experiments in monkeys, a relatively low clinical dosage was
set at about 10 mg per adult, per day (while that of enfuvirtide
was 180 mg[9]). The PK profiles and bioavailability of
sifuvirtide following single sc or iv dosage of 1.2 mg/kg in
rhesus monkeys were investigated. The preliminary results
indicated that sifuvirtide made a difference in antiviral
activity in vitro and PK behaviors in
vivo compared with enfuvirtide.
Materials and methods
Drugs and reagents The sifuvirtide standard was
provided by FusoGen Pharmaceuticals (Tianjin, China).
Enfuvirtide was obtained from Hoffmann-La Roche (Nutley,
NJ, USA). RPMI-1640 complete culture medium containing
10% heat-inactivated newborn calf serum (NCS),
L-glutamine 2 mmol/L, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid (HEPES) 10 mmol/L, 2-mercaptoethanol 50 µmol/L,
benzylpenicillin 100 kIU, and streptomycin sulfate 100 mg/mL
were purchased from Gibco BRL (Gaithersburg, Maryland,
USA) . C8166 and HIV-1IIIB chronically infected H9 cell lines
(HIV-1IIIB/H9) were kindly donated by the Medical Research
Council of United Kingdoms (AIDS Reagent Project) (London, UK). Drug-free monkey plasma was collected from
6 healthy rhesus monkeys of both sexes. Acetonitrile (ACN)
and other solvents were of HPLC-grade and purchased from
Fisher Scientific (Pittsburgh, PA, USA). A protease inhibitor
cocktail tablet, Complete Mini (Roche Diagnostics,
Mannheim, Germany), without ethylene diamine tetraacetic
acid, was used to prevent peptide degradation in biological
matrixes. Formic acid (FA), trifluoroacetic acid (TFA) and
sodium iodine were obtained from Aldrich Chemical Co
(Milwaukee, WI, USA). Highly purified water was produced
by a Millipore Simplicity 185 Unit (Millipore, Bedford, MA,
USA). Mobile phase A (MPA) was 0.1% (v/v) FA in water.
Mobile phase B (MPB) was 0.1% (v/v) FA in ACN.
Synthesis of the internal standard In this study, a novel
strategy, non-radioactive iodination of peptides, was used
to acquire an internal standard (IS) for the quantitative assay.
Reaction of iodination was carried out mainly as described
previously[11]. To acquire the highest productivity of
peptide iodide, the molar ratio of iodine-127 to peptide standard
was at least 20:1. The reaction was carried out at room
temperature for 10 min. The reaction mixture was then desalted
and purified by a RP-HPLC method utilizing a Vydac
analytical C18 column (5 µm, 300 Å, 4.6 mm×150 mm; Vydac, Hesperia,
CA, USA) with a gradient of MPB increasing from 25% to
75% in 20 min, coupled with fraction collection equipment
(Pharmacia, Uppsala, Sweden). The purified product was
stored at 4 °C and identified using LC/MS/MS to be the goal
peptide (purity >97%) with the 4-127I modifications
(127I4-sifuvirtide) on tyrosine (Y) residues
[SWETWEREIENY(I2)TRQIY(I2)RILEESQEQQDRNERDLLE].
Cell fusion assay The inhibition of sifuvirtide on
cell-to-cell transmission of HIV-1 was determined by co-cultivation
of uninfected normal cells and
HIV-1IIIB chronically infected cells, as described
previously[12]. Briefly,
3×104 C8166 cells were mixed with
1×104 HIV-1IIIB chronically infected H9 cells
in 96-well plates in the presence or absence of sifuvirtide at
various concentrations. Enfuvirtide was used as the control.
After co-culture for 24 h at 37 ºC in 5%
CO2, the number of syncytia was counted using an inverted microscope. The
percentage inhibition of cell fusion was calculated by
dividing the number of syncytial cells in compound-treated
cultures by those in untreated cultures. The
IC50 was determined from a dose-response curve.
On-line sample preparation The on-line extraction
column was prepared using an empty stainless steel column
(size of 50 mm length×2.1 mm ID, 2 µm frit; GEA Co, Beijing,
China) packed with reversed-phase materials. The packing
materials used for SPE were obtained from Agilent (Zorbax
C18; spherical; average particle size 50 µm; pore size 80 Å;
Agilent; Waldbronn, Germany). Aliquots of calibrators or
unknown samples (100 µL) were introduced onto the
extraction column for on-line preparation and LC/MS/MS analysis.
The plasma samples were diluted 5 times on-line, based on
the design of an on-line splitter, between the sample loop
and bypassing branch at a ratio of approximately 1:4. The
whole process, including sample loading, extraction,
separation and analysis, was carried out based on the
column-switching program. Programmed events corresponding to
valve switching were set up using the ChemStation (version
9.02) for Agilent 1100 series HPLC system and
XcaliburTM (version 1.4) for LTQ-MS system.
LC/MS/MS system The separation and detection
section, the LC/MS/MS system used for the assay, was made
up of the Agilent 1100 series HPLC system (including an
on-line degasser), a 100-vial autosampler, a temperature control
compartment equipped with a 6-port/2-position switching
valve, and a binary solvent delivery pump, coupled to a novel
linear ion trap mass spectrometer, LTQ-MS (Thermo Finnigan,
San Jose, CA, USA), equipped with a new atmospheric
pressure ionization interface, Ion MAX™. MS/MS detection
was operated in electrospray ionization positive ion mode.
The representative parameters of MS were as shown below.
The spray voltage was 4.5 kV and the temperature of the
heated capillary was set at 200 °C. The flow rates of sheath
gas, auxiliary gas and sweep gas were set (in arbitrary
units/min) to 20, 5, and 4, respectively. Other parameters
were optimized automatically by infusing the analyte in
water:ACN:formic acid (49.9:50:0.1, v:v:v) at a flow rate of 200µL/min. Quantitation was carried out using selected
reaction monitoring (SRM) of the transitions
m/z 946.5® m/z 871.8 for sifuvirtide and
m/z 1047.2®m/z 972.3 for the IS
(127I4-sifuvirtide).
Preparation of stock solutions and calibration standard
samples Stock solutions of sifuvirtide were prepared at
concentrations of 1.0 g/L in a
MPA:MPB (90:10, v/v) solvent
system. The drug-free monkey blood was deactivated by
spiking with protease inhibitor cocktail tablets (2
tablets/5 mL whole blood). All blood samples collected from
monkey sources were transferred immediately into tubes coated
with calculated heparin and an inhibitor mixture of protease
and peptidase on the tube surface. After incubation on ice
for 30 min, they were centrifuged at
1000×g for 10 min. The supernatant of each was then harvested and stored at -80 °C
until analysis. The deactivated drug-free plasma from
monkey sources was used as the standard dilution for the
calibration samples. Each calibration curve consisted of blank
samples, zero samples (plasma sample processed with IS),
and 6 non-zero samples, ranging from 4.88 µg/L to 5000
µg/L. Before the assay, the frozen samples were thawed at ambient
temperature, vortex-mixed and centrifuged at
1000×g for 5 min. For each of the calibrators or unknown samples, 100 µL
aliquots of supernatant were added to vials containing 10 µL
127I4-sifuvirtide working solution (5 mg/L) and 10 µL 2.5%
ammonia solution
(NH3·H2O, w/v). The vials were
vortex-mixed for 30 s, placed immediately in the autosampler tray
and kept at room temperature for assay.
Method validation The calibration curve and the
concentration of unknown sample were acquired using LCQuan
software (version 2.0) from Thermo Finnigan. A weighting
of 1/x2 (where x is the concentration of a given standard)
was used for curve fit. The intra-day and inter-day accuracy
and precision of the method presented here was
investigated by analyzing plasma samples at the whole calibration
concentration levels. The regression equation for the
calibration curve was used to back-calculate the measured
concentration for each standard, and the results were compared
to the theoretical concentration to calculate the accuracy,
expressed as a percentage of the theoretical value, for each
standard measured.
Animal experiments Three rhesus monkeys
(2¡â and 1¡á, body weight of 4.1±0.4 kg) were provided by the Animal
Center of the Academy of Military Medical Sciences, with
certificate number 97083. The monkeys were fasted
overnight prior to dosing and given free access to food and
water 48 h post dosage. A dosing solution (1.0 g/L) was
formulated in saline. Sifuvirtide was administered iv to monkeys at
1.2 mg/kg via a side femoral venous catheter. Blood samples
(0.5 mL) were collected from a femoral venous catheter on
the opposite side at 0 min, 1 min, 5 min, 15 min, 30 min, 1 h,
2 h, 4 h, 6 h, 8 h, 12 h, 24 h, and 48 h after dosing. After a
7-d washout period, the monkeys were administered the same
dosage and blood samples were harvested at 0 min, 10 min,
15 min, 30 min, 1 h, 2 h, 4 h, 5 h, 6 h, 8 h, 10 h, 12 h, 24 h, 36 h,
and 48 h following dosing. All samples were harvested and
treated in the same way as calibrators. A 200 µL aliquot from
each time point was used for the assay. The plasma samples
from 1 min, 5 min, and 15 min after iv were diluted 5 times
because their concentrations were suspected to be higher
than detectable levels. The curve according to plasma
sifuvirtide concentration versus time after dosing was
obtained using Origin software (Version 5.0; Microcal,
Northampton, MA, USA).
Estimation of pharmacokinetic parameters
Pharmacokinetic modeling and estimation of PK parameters were
carried out using Excel XP software (Microsoft, Redmond, WA,
USA). Cmax and
Tmax were observed values. Other PK
parameters, including area under the concentration-time
curve (AUC), apparent plasma terminal elimination
T1/2, mean residence time (MRT), volume of distribution
(Vd) and systematic clearance (CL), were calculated according to a
previous description of the non-compartmental
approach[13]. In addition, the absolute bioavailability
(Fabs, %) was calculated by the following equation: [(subcutaneous
AUC0-t/intravenous
AUC0-t)×100%].
Results
Effects of sifuvirtide on HIV-1 entry Sifuvirtide
inhibited syncytium formation between
HIV-1IIIB/H9 cells and C8166 cells with an
IC50 value of 0.33 µg/L. The
IC50 of sifuvirtide was approximately 50-fold lower than that of
enfuvirtide (16.8 µg/L). The results suggested that sifuvirtide
was more effective than enfuvirtide in blocking HIV-1 entry
into cells.
Mass spectrometry detection of sifuvirtide and
127I4-sifuvirtide
The mass spectrum obtained by infusing
10 mg/L solution of sifuvirtide and
127I4-sifuvirtide in water:ACN
(50:50, v/v) at a flow rate of 5 µL/min is shown in Figure 1A. A
series of multiply charged ions,
[M+5H]5+ at m/z 946.5,
[M+4H]4+ at m/z 1182.9 and
[M+3H]3+ at m/z 1576.6 for sifuvirtide, and
[M+5H]5+ at m/z 1047.2,
[M+4H]4+ at m/z 1308.7 and
[M+3H]3+ at m/z 1744.6 for
127I4-sifuvirtide, were observed
in positive ion mode. Deconvoluted mass obtained
automatically utilizing the Bioworks software (Ver 3.1 SR1,
Thermo-Electron, San Jose, CA, USA) showed a difference
of 503.3 u (theoretical value was 503.6 u) between sifuvirtide
and 127I4-sifuvirtide, which demonstrated that a complete
iodination had been performed and reached the goal product.
The most abundant ions of sifuvirtide and
127I4-sifuvirtide,
[M+5H]5+ at m/z 946.5 and m/z
1047.2, respectively, were selected as parent ions for a collision-induced dissociation
experiment. The most intensive product ions for sifuvirtide
and IS were observed at m/z=871.87
(b335+) and m/z=972.6
(b335+), respectively, both with optimized normalized
collision energy of 28%[10].
Method validation Under the established
SPE-LC/MS/MS conditions, the typical retention times for sifuvirtide and
IS were 7.68 min and 7.91 min, respectively, without
interference from endogenous plasma components. The limit of
detection was 1.22 µg/L, which represented 122 pg
on-column injected analyte (approximately 26 fmol in drug-free
monkey plasma, absolute recovery was seen as 100%), with
a signal to noise (S/N) ratio above 10. The typical
chromatograms of sifuvirtide in monkey plasma are illustrated in
Figure 2. Calibration curves exhibited excellent linearity over
a range of 4.88 mg/L to 5000 µg/L, and the typical regression
equation for sifuvirtide was
y=0.01736-0.003759x, with a
r2 of 0.9929. The intra-assay and inter-assay precision and
accuracy values of the method were assessed using spiked
plasma samples at whole calibration concentration levels for
sifuvirtide, shown in Table 1. The mean accuracy of
back-calculated concentrations of the standards compared with
theoretical ones ranged from -5.36% to 4.61%. The
inter-batch CV ranged from 2.9% to 8.8%, and the intra-batch CV
did not exceed 10.5%. The stability of sifuvirtide in solution
and in plasma samples was also
evaluated[10]. The areas ratios of SRM chromatography spectra of sifuvirtide versus
IS were investigated in stock solution kept in 4 °C for
approximately 15 d and in plasma samples left at 15 °C
overnight (12 h-14 h). No real change in the area ratio of sifuvirtide
to IS was observed.
Phamacokinetics of sifuvirtide in rhesus
monkeys The individual concentration-time profiles of sifuvirtide after iv
or sc administration are shown in Figure 3. Plasma
concentration versus time data was analyzed and the PK parameters
are shown in Table 2. After administration of sifuvirtide at
doses of 1.2 mg/kg, the observed peak concentration was
10 626±2886 µg/L for iv at 1 min, and 528±191 µg/L for sc at
0.25 h-2 h, respectively. The terminal elimination plasma
half-lives (T1/2) after iv or sc were 6.3±0.9 h and 5.5±1.0 h,
respectively. Following sc administration, sifuvirtide was
absorbed and distributed rapidly, and meanwhile was
eliminated quickly in plasma. The bioavailability after sc
injection was 49%±13%. In the present study, a remarkable
inter-individual variability of the PK parameters, such as AUC,
Cmax, Tmax, and
Fabs, of sifuvirtide was observed in both iv
and sc administration groups.
Discussion
In the present study, the advanced on-line
SPE-LC/MS/MS system was validated as being reliable, reproducible,
and highly sensitive for the quantitative determination of
sifuvirtide in monkey plasma. The programmable column-
switching technique was the core of this fully automatic
system. Based on the design of on-line splitting injection
mode (OSIM), this novel on-line SPE system showed a
powerful performance and capability for sample preparation of
more than 300 raw plasma samples. The addition of pH
adjusting solvent is very important for increasing the
extraction efficiency of sifuvirtide. Coupled with LTQ-MS, a novel
quadrupole ion trap mass spectrometer, a satisfactory
sensitive and good calibration curves yielding a wide linearity
range over 3 orders of magnitude with correlation
coefficients higher than 0.9923 were obtained.
Due to the lack of effective vaccines for the prevention
of HIV infection, development of potent and affordable
anti-HIV drugs is an international priority. As the first fusion
inhibitor, enfuvirtide has been considered to have the
disadvantages of short elimination half-life (2.5 h-3.5 h in the
monkey via sc route[14]), which is administered twice at a
high cost. A lower IC50 value of sifuvirtide for cell-to-cell
transmission inhibition of HIV-1
in vitro was determined at around 0.33 µg/L, which is lower than that of enfuvirtide
(16.8 µg/L). Therefore, a relatively lower dosage of sifuvirtide
is recommended for future clinical treatments. In addition,
the PK parameters of sifuvirtide after sc or iv dosing
at 1.2 mg/kg weight in rhesus monkeys were below those of
enfuvirtide (0.8 mg/kg in cynomolgus
monkey[14]). Although the average observed concentration of sifuvirtide at 10 min
after dosing was lower than that of enfuvirtide, the plasma
concentration profiles of sifuvirtide after iv or sc were
sustained at a relatively higher ratio to the
in vitro IC50, even at 36 h after dosing. The terminal half-life of sifuvirtide was
estimated as 5.5 h-7.2 h after iv and 4.6 h-6.6 h after sc
administration, which is longer than that of enfuvirtide. A
lower average AUC0-¥ and higher mean
Vd and CL (normalized by animal weight) was found for sifuvirtide compared with
enfuvirtide, which indicated that sifuvirtide was widely
distributed to tissues or organs via circulation of blood and
showed the benefit of antiviral action
in vivo. These results were in accordance with those derived from tissue
distribution experiments by radioactive determination (data not
shown). The comparative MRT and Fabs (%) of two peptides
were estimated in monkeys by the non-compartmental
approach. All of these results derived from inhibitory
activity in vitro and PK characteristics provide an important
reference for future clinical studies.
Acknowledgements
We thank Dr Zhi-yun MENG and Mr Jun LI for their
helpful discussions and excellent technical support.
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