500
GU ET AL.
diluted with saturated NaCl (100 ml) and then extracted with EtOAc; the
Excretion of Radiolabeled PR-104 in Mice. Mice were transferred to glass
organic extract was washed with saturated NaCl, dried, and evaporated. The metabolic cages (Minor Metabowl, Jencons, UK; one animal per cage) 2 h
residue was purified by chromatography on silica gel, eluting with 5% MeOH/
before treatment and had continuous access to food and water before and
EtOAc, and combined early fractions were concentrated and diluted with during experiments. [3H]PR-104 was administered intravenously via a lateral
i-Pr2O to give 2-[2-[[(2-hydroxyethyl)amino]carbonyl]-4,6-dinitroanilino]- tail vein at 326 mg/kg. Urine was collected at 4, 8, 24, and 48 h. Metabolic
ethyl methanesulfonate (M8) (89 mg, 27%): mp 122°C; 1H NMR [(CD3)2SO] cages were rinsed with 2 ml of water into the urine sample to minimize
␦ 9.11 (t, J ϭ 5.3 Hz, 1H), 8.98 (t, J ϭ 5.4 Hz, 1H), 8.84 (d, J ϭ 2.7 Hz, 1H), carryover between time points. Feces were collected at 24 and 48 h.
8.41 (d, J ϭ 2.7 Hz, 1H), 4.79 (t, J ϭ 5.5 Hz, 1H), 4.43 (t, J ϭ 5.0 Hz, 2H),
3.59–3.51 (m, 4H), 3.34 (q, J ϭ 5.7 Hz, 2H), 3.19 (s, 3H). Elemental analysis
Determination of Total Radioactivity and Radioactivity Profiles. Radio-
activity was determined using a liquid scintillation analyzer (Tri-Carb 1500;
calculated for C12H16N4O9S: C, 36.74; H, 4.11; N, 14.28; S, 8.17; found: C, PerkinElmer Life and Analytical Sciences, Waltham, MA), with samples
37.11; H, 4.29; N, 14.17; S, 8.20.
PR-104S2 (M10). A suspension of 4 (150 mg, 0.51 mmol) in 24 wt.%
aqueous HBr (6 ml) was stirred at room temperature for 3 h, then diluted with
counted for 10 min unless 2 of 0.4% was reached. An external standard
quench curve was used to correct for differences in counting efficiency. Urine
samples for total radioactivity measurement were diluted into water-accepting
water (25 ml), and extracted with EtOAc (2 ϫ 20 ml). The combined extract scintillation mixture (Emulsifier-Safe; PerkinElmer Life and Analytical Sci-
was dried, filtered through a plug of silica gel, concentrated to a small volume ences, Waltham, MA). Feces were dried, powdered, and rehydrated by addition
under reduced pressure, and then diluted with i-Pr2O to give 2-[(2-bromoethyl)- of deionized water. One milliliter of Soluene-350 (PerkinElmer Life and
amino]-N-(2-hydroxyethyl)-3,5-dinitrobenzamide (M10) (177 mg, 93%): mp Analytical Sciences) was added and incubated at 37°C overnight; 0.5 ml of
122 to 123°C; 1H NMR [(CD3)2SO] ␦ 9.09 (t, J ϭ 5.4 Hz, 1H), 8.97 (t, J ϭ isopropyl alcohol was added; and samples were incubated for 12 h at 37°C.
5.5 Hz, 1H), 8.85 (d, J ϭ 2.8 Hz, 1H), 8.38 (d, J ϭ 2.7 Hz, 1H), 4.80 (t, J ϭ
5.5 Hz, 1H), 3.75 (t, 5.9 Hz, 2H), 3.63 (q, 5.8 Hz, 2H), 3.56 (q, 5.8 Hz, 2H),
Next, 0.2 ml of 30% H2O2 was added drop-wise with swirling to bleach. After
standing for 10 min at ambient temperature, samples were warmed to 37°C for
3.34 (q, J ϭ 5.8 Hz, 2H). Elemental analysis calculated for C11H13BrN4O6: C, 15 min to decompose peroxides and thus minimize chemiluminescence. Ten to
35.03; H, 3.47; N, 14.86; Br, 21.19; found: C, 35.20; H, 3.54; N, 14.95; Br,
21.04.
15 ml of scintillation fluid (Hionic-Fluor; PerkinElmer Life and Analytical
Sciences) was added. For quantification of radioactivity in HPLC eluents,
Subjects. Specific pathogen-free homozygous nu/nu (CD1-Foxn1nu) mice fractions were collected at 0.1-min intervals using an Agilent 1100 auto-
and Sprague-Dawley rats (Charles River, Margate, Kent, UK) were bred in the fraction collector (Agilent Technologies, Santa Clara, CA), transferred to
University of Auckland. Mice were housed in Tecniplast (Buguggiate, Italy)
microisolator cages in groups of four to six in a temperature-controlled room
(20 Ϯ 2°C) with a 12-h light/dark cycle and were fed ad libitum UV-treated
Milli-RO water (Millipore Corporation, Billerica, MA) and a sterilized rodent
diet (diet 2018s; Harlan Teklad, Madison, WI). Rats were housed in groups of
four to six under the same conditions but received filtered tap water and diet
2018 (Harlan Teklad). At the time of experiments, animals weighed 25 to 30 g
(mice) and 200 to 220 g (rats). The rodent studies were approved by the
University of Auckland Animal Ethics Committee. Beagle dogs (7–8 months
of age, 10–13 kg) were from Kangda Laboratory Animals S and T Co., Ltd.
(Gaoyao, China). The dog study was conducted at LAB PreClinical Research
International Inc. (Laval, QC, Canada) and approved by the Institutional
Animal Care and Use Committee. The dogs were individually housed in a
room maintained at 21 Ϯ 3°C and fed Teklad Certified Canine Diet (8727C;
Harlan Teklad) with water ad libitum. A 12-h light/dark cycle was maintained.
scintillation vials, mixed with Emulsifier-Safe, and counted for radioactivity.
Metabolite Profiling. Urine samples were diluted 1:10 with water, filtered
through 0.22-m filters, and analyzed directly by HPLC with photodiode array
absorbance and MS detection (Agilent 1100 LC/MSD model A; Agilent
Technologies). Mouse bile samples (gallbladder) were precipitated with ap-
proximately 10 volumes of methanol, centrifuged, and diluted into formate
buffer. The chromatographic separation was performed on an Alltima C8
analytical column (150 ϫ 4.6 mm, 5 m; Alltech Associates, Deerfield, IL)
with a flow rate of 0.7 ml/min maintained at 25°C. The mobile phase was an
acetonitrile gradient constructed using 80% acetonitrile/20% water v/v (A) and
45 mM ammonium formate buffer in water at pH 4.5 (B) with 20% of A for
2 min and then increasing linearly to 80% A over 13 min, held for a further 5
min, returned to the initial composition over 2 min, and maintained for 5 min
before the next injection. Absorbance detection was at 370 nm (bandwidth, 4
nm). An Agilent LC/tandem MS (MS/MS; model 6410) equipped with an
Human subjects, from two Phase I trials, were patients with a pathologically electrospray ionization/atmospheric pressure chemical ionization multimode
confirmed solid malignancy not amenable to standard therapy, age Ն18 years, source was used for further identification of some products in excretion
Karnofsky performance status Ն70%, adequate renal and liver function, and samples.
Ͼ4 weeks since previous surgery, radiotherapy, or chemotherapy.
Mouse and rat plasma samples were prepared by precipitating proteins with
Dosing and Sample Collection. PR-104 free acid was dissolved in phos- 3 volumes of methanol, and for dog and human plasma with 9 volumes of
phate-buffered saline ϩ1 equivalent NaHCO3 and diluted in phosphate-buff- acidified methanol (methanol/ammonium acetate/acetic acid, 1000:3.5:0.2,
ered saline (rodents and dogs), or the clinical formulation (PR-104 sodium salt
lyophilized with mannitol) was reconstituted in 2 ml of water and diluted in 5%
dextrose (humans). Dosing was by the intravenous route in all the cases. The
v/w/v) as described previously (Patel et al., 2007). Precipitated samples were
diluted into 2 volumes of purified water and analyzed by the LC/MS method
reported previously (Patel et al., 2007). In brief, a 150 ϫ 2.1-mm column with
[3H]PR-104 (free acid) was similarly titrated with NaHCO3 and diluted to a flow rate of 0.3 ml/min was used, with positive and negative mode atmospheric
specific activity of 2.85 GBq/mmol (or 77.0 mCi/mmol) with unlabeled
PR-104. Mice were dosed at 326 mg/kg (975 mg/m2) via a lateral tail vein and
pressure electrospray ionization. The mass/charge (m/z) ratio was scanned
from 200 to 800 with fragmentor voltage of 100 V. An Agilent LC/MSD
sampled by cardiac puncture after cervical dislocation. Gallbladders were trap-SL ion trap mass spectrometer equipped with an Agilent capillary HPLC
collected from four mice 30 min after dosing at 326 mg/kg. Rats were dosed
at 244 mg/kg (1450 mg/m2) via a tail vein and bled serially from the saphenous
veins for up to 3 h. Urine was collected at intervals of 1 h for up to 3 h from
four rats. Dogs were dosed into the cephalic or saphenous vein as a slow bolus
injection at 150 mg/kg (3000 mg/m2), and blood samples were collected from
the jugular vein for up to 2 h. Patients were dosed as a 1-h infusion, and plasma
and urine samples were collected after the first dose of a once per 3-week
schedule. Urine samples were collected over 24 h after a range of doses
(135–1400 mg/m2) at a single clinical site (Hamilton, NZ), and blood samples
system was used for further identification of some metabolites using a Zorbax
SB C18 capillary column (150 ϫ 0.5 mm, 5 m; Agilent Technologies) at a
flow rate of 15 l/ml (Patel et al., 2007). The electrospray ionization source
was set at positive ionization mode with auto MS(n).
Hepatic 9000g Postmitochondrial Supernatant Metabolism of PR-104A. In
vitro hepatic metabolism of PR-104A was studied using liver 9000g postmi-
tochondrial supernatant (S9) fraction prepared from pooled CD-1 nu/nu mice
in-house and pooled human liver S9 purchased from CellzDirect (Durham,
NC). Reactions (final volume, 0.1 ml/well in 96-well plates) comprised hepatic
were collected over 5 h (from start of infusion) from six patients at the S9 (2 mg of protein/ml), PR-104A (150 M), and cofactor (NADPH, NADH,
maximum tolerated dose (1100 mg/m2) (Jameson et al., 2009). In addition,
blood was collected from four patients at the same dose level in a second Phase I
study after the first dose of a weekly dosing schedule. All the blood samples were
collected into tubes containing K2EDTA. These samples were placed on ice
immediately after collection and centrifuged within 10 min to harvest plasma.
or both, 1 mM each) in sodium/potassium phosphate buffer (67 mM, pH 7.4)
with 5 mM MgCl2 and 1 mM EDTA and were incubated for 30 min at 37°C
under air or in an anaerobic chamber (Sheldon Manufacturing, Cornelius, OR).
Incubations with boiled S9 preparations were used as controls. All the solu-
tions for the anoxic experiment were equilibrated (along with the 96-well
Gu, Yongchuan
