VOL. 48, 2004
PRECLINICAL DEVELOPMENT OF GW433908
793
Louis, Mo.). Within 10 min, a white precipitate was observed. The material was
collected by filtration and identified as APV by spectroscopic and chromato-
graphic comparison with a commercial sample.
mination of plasma APV concentrations. Each 2.5-ml whole-blood sample was
obtained from the cephalic catheter and collected into a sodium citrate-contain-
ing glass Vacutainer tube. Plasma was separated by refrigerated centrifugation
and stored frozen at Ϫ20°C until analyzed.
Historical APV pharmacokinetic data for the same dogs were used to deter-
mine relative bioavailability. Doses of APV (300 mg in vitamin E-TPGS [D-alpha
tocopherol polyethylene glycol 1000 succinate), polyethylene glycol 400, and
propylene glycol) were administered orally in two soft-gelatin capsules. Samples
were collected and handled as described above.
(ii) GW433908 portal vein sampling study. A single dose of an oral suspension
of the calcium salt of GW433908 (28.0 mg/ml; 22.8 mg of free acid/ml) in 0.5%
hydroxypropylmethylcellulose (prepared in 0.1% Tween 80) was administered by
gavage to seven male Han Wistar rats and one male beagle dog for portal vein
sampling. The rats were divided into three groups with each group having
different blood collection times as described below. Prior to dosing, the dog was
administered 100 ml of 0.05 N HCl solution to produce a favorable gastric
environment for GW433908 calcium salt dissolution. Rats received a single dose
of 112 mg of GW433908 calcium salt/kg of body weight (91.3 mg of GW433908
free acid/kg, 4 ml/kg), and the dog received a single dose of 35 mg of GW433908
calcium salt/kg (28.5 mg of GW433908 free acid/kg, 1.25 ml/kg).
For determination of GW433908 free acid and APV concentrations, serial
whole-blood samples (0.5 ml) were collected from rats in tubes containing so-
dium citrate according to the following composite schedule: t ϭ 0 (predose), 1.0,
4.0, and 12.0 h (group 1); t ϭ 0.25, 2.0, 6.0, and 24.0 h (group 2); and t ϭ 0.50,
3.0, and 8.0 h (group 3). Eleven serial whole-blood samples (1.8 ml) were
collected from the dog over 24 h (t ϭ 0 [predose], 0.25, 0.50, 1.0, 2.0, 3.0, 4.0, 6.0,
8.0, 12.0, and 24.0 h). Each whole-blood sample was obtained from the portal
vein cannulae and collected into sodium citrate-containing glass Vacutainer
tubes. Plasma was separated by refrigerated centrifugation and stored frozen at
Ϫ20°C until analyzed.
Assays for concentrations of APV and GW433908 free acid in plasma. Plasma
APV concentrations after administration of APV and the free acid and calcium
salt formulations of GW433908 (study 1) were analyzed with a validated method
with fluorescence detection (excitation at 245 nm, emission at 340 nm). APV was
extracted from thawed plasma samples (0.5 ml of plasma combined with 0.5 ml
of internal standard solution) by solid-phase extraction with a Waters MilliLab
Workstation and C18 Sep-Pak cartridges. After extraction, 50-l samples were
injected onto a Waters Symmetry C18 chromatography column (150 by 3.9 mm)
at 40°C. Samples were eluted from the column with a mobile phase of 43%
acetonitrile in water (43:57, vol/vol) at a constant flow rate of 0.8 ml/min.
Samples were spiked with an internal standard solution (VB 11599, 5 g/ml;
Vertex Pharmaceuticals). Peak areas for APV and internal standards, as well as
linear regression of the standards, were determined with HP 1090 ChemStation
software version A.03.01. APV concentrations in control and unknown standards
were determined from the regression curve (1/C2 weighting). The lower limit of
quantitation (LLOQ) for the assay was 0.05 g/ml. Recovery of APV with this
method was 86 to 94%, and recovery of the internal standard was 91 to 98%.
Estimates of intra-assay precision (percent coefficient of variation [CV]) and
accuracy (percent bias) determined with spiked validation rat control samples for
APV were 1.1 to 4.4% and Ϫ4.2 to Ϫ1.9%, respectively. Estimates of intra-assay
precision (percent CV) and accuracy (percent bias) determined with spiked
validation dog control samples for APV were 1.4 to 5.6% and Ϫ3.5 to 8.2%,
respectively.
APV and GW433908 concentrations in plasma following oral administration
of the sodium salt formulation of GW433908 (study 1) were determined by
reversed-phase HPLC analysis with tandem mass spectrometric detection
(HPLC-MS-MS) with an HP1100 HPLC system interfaced to a Sciex API-365
triple quadrupole mass spectrometer (MDS Sciex, Concord, Ontario, Canada).
Plasma samples (100 l) were deproteinated by addition of 500 l of ethanol-
acetonitrile (1:1, vol/vol) followed by centrifugation (15,800 ϫ g, 4°C, 5 min). The
resulting supernatants were evaporated to dryness, and the dried extracts were
reconstituted to the original sample volume with 20% acetonitrile in 0.1% aque-
ous acetic acid adjusted to pH 5.4 with ammonium hydroxide (initial mobile
phase). The plasma extracts (10 l) were injected onto a Luna reversed-phase
C18 column (100 by 2.0 mm, 5 m; Phenomenex) at room temperature. Samples
were eluted with a 5-min linear gradient from 20 to 95% acetonitrile in 0.1%
acetic acid, pH 5.4, followed by a 4-min isocratic elution with 95% acetonitrile–
0.1% acetic acid, pH 5.4, at a constant flow rate of 0.2 ml/min. GW433908 and
APV eluted at approximately 6.5 and 7.0 min, respectively, and were detected by
positive-ion multiple reaction monitoring, monitoring the transitions m/z 586.3 to
m/z 418.2 and m/z 506.3 to m/z 245.2, respectively, at a collision energy of 30 eV.
Comparison of the nominal standard concentrations to the peak areas was
performed with 1/C2-weighted calibration curves generated with the MacQuan
Dependence of GW433908 solubility on pH. The aqueous solubility of
GW433908 was determined for the free acid (GW433908X), bis-sodium salt
(GW433908A), and calcium salt (GW433908G) early in the development pro-
cess. Only single points were assayed per pH-solvent, and quantification was
performed with the UV spectrometric assay, described below. For GW433908A
(bis-sodium) solubility was estimated by visual inspection. The aqueous solubility
of the calcium salt (GW433908G) was determined in duplicate experiments.
Samples were assayed three or four times over 10 to 18 days to demonstrate
equilibrium, with the average of the last two pHs and solubility values reported.
Quantification was performed with
a high-pressure liquid chromatography
(HPLC) assay as follows: solubilities were determined by equilibrating excess
solid in the solvent of interest at 25°C. Solid was separated from solution by
filtration with Gelman 0.45-m-pore-size nylon filters (UV assay) or Millipore
0.45-m-pore-size polyvinylidene fluoride filters (HPLC assay). For the UV
assay, samples were diluted in pH 9 borate buffer to approximately 0.05 mg/ml
and absorbances were measured using a Perkin-Elmer Lambda 18 UV-visible
light spectrophotometer. Sample concentrations were calculated using a stan-
dard curve of the drug in pH 9 borate buffer. For the HPLC assay, samples were
diluted in methanol to approximately 0.2 mg/ml and injected onto a Phenomenex
Luna C18 (2) chromatography column (50 mm by 2 mm; 3 m) at 40°C. Samples
were eluted from the column using a gradient mobile phase (A, 0.05% triflu-
oroacetic acid in water; B, 0.05% trifluoroacetic acid in acetonitrile; gradient 0%
B to 95% B over 8 min with a 2-min reequilibration). Sample concentrations
were analyzed using a Hewlett-Packard HP1100 HPLC chromatograph with UV
detection (220 nm), with peak areas compared to that of an external standard.
The negative logarithms of the equilibrium constants (pKs) of GW433908
were determined as follows: pK1 and pK2 were calculated by using a least-
squares model in the program Micromath Scientist for Windows (version 2.01) to
fit the solubility data for the pH range 0.4 to 3.3; pK3 and pK4 were calculated
with the ACD/Labs pKa DB (version 3.0) software.
Animals. Male beagle dogs, weighing 10 to 14 kg, were purchased from Mar-
shall Research Animals (North Rose, N.Y.). One dog had a portal vein cannula
surgically implanted. Male Han Wistar rats, weighing 0.22 to 0.26 kg, were
purchased from Taconic Farms (Germantown, N.Y.). Rats were acclimated for
1 week prior to surgical placement of a portal vein cannula and allowed to
recover from surgery for at least 36 h prior to dosing. Dosing and blood collec-
tion were conducted at GlaxoSmithKline (International Development BioMe-
tabolism, Research Triangle Park, N.C.).
The protocol and any amendment(s) or procedures involving the care and use
of animals in these studies were reviewed and approved by GlaxoSmithKline’s
Institutional Animal Care and Use Committee prior to the experiment. During
the study, the care and use of animals were performed in accordance with the
guidelines of the U.S. National Research Council.
Pharmacokinetic studies. (i) APV exposure and bioavailability from clinical
formulation and prodrug formulations. A crossover study design, with at least a
7-day washout period between doses, was used to administer single doses of APV
and four oral preparations of GW433908 to three male beagle dogs. Animals
were weighed and fasted for 16 h before and 8 h after each dose.
Four GW433908 formulations were tested: (i) hard-gelatin capsules containing
the free acid of GW433908, (ii) hard-gelatin capsules containing the bis-sodium
salt of GW433908, (iii) tablets containing the calcium salt of GW433908 without
citrate (administered on two separate occasions), and (iv) the calcium salt with
citrate. The free acid of GW433908 (60%) was combined with lactose (35.7%),
Ac-Di-Sol (croscarmellose sodium; 3.3%), and sodium lauryl sulfate (1.0%). The
bis-sodium salt capsules did not contain excipients. Two compressed tablet prep-
arations containing the calcium salt of GW433908 were synthesized. In prepa-
ration A, the calcium salt (56.7%) was combined with Avicel (microcystalline
cellulose; 39.4%) and crospovidone (3.9%). In preparation B, the calcium salt
(56.4%) was combined with Avicel (29.7%), crospovidone (4.0%), and citric acid
(10%). The dogs received two hard-gelatin capsules containing the free acid
(total GW433908 free acid dose, 360 mg), one hard-gelatin capsule containing
the sodium salt (total GW433908 sodium salt dose, 250 mg), and two tablets
(without and with citric acid) containing the calcium salt (total GW433908
calcium salt dose, 418 and 434 mg, respectively). Except for the sodium salt form
(at ca. 220 mg of APV equivalent dose), the GW433908 dose of each formulation
was equivalent to approximately 300 mg of APV. Immediately prior to the repeat
dosing with tablets containing the calcium salt of GW433908 formulated without
citric acid, the dogs were administered 100 ml of 0.05 N HCl (pH 1.7) by gavage.
A total of 11 serial whole-blood samples were collected over 24 h (t ϭ 0
[predose], 0.25, 0.50, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, 12.0, and 24.0 h) for the deter-