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Mao J, Xu Y, Wu D and Almassain B
Pharmacokinetics, Mass Balance, and Tissue Distribution of a Novel DNA Alkylating Agent, VNP40101M, in Rats
AAPS PharmSci
2002;
4
(4)
article 24
( https://www.aapspharmsci.org/scientificjournals/pharmsci/journal/ps040424.htm
).
Pharmacokinetics, Mass Balance, and Tissue Distribution of a Novel DNA Alkylating Agent, VNP40101M, in Rats
Submitted: April 22, 2002; Accepted: June 30, 2002; Published: October 7, 2002
John Mao
1
, Yang Xu
1
, Diana Wu
2
and Bijan Almassain
1
1
Development, Vion Pharmaceuticals, Inc, Four Science Park, New Haven, CT 06511
2
Metabolism, XenoBiotic Laboratories, Inc, 107 Morgan Lane, Plainsboro, NJ 08536
|
Correspondence to:
John Mao
Telephone:
Facsimile: (203) 498-4211
E-mail: jmao@vionpharm.com
|
Keywords:
VNP40101M
Mass balance
Pharmacokinetics
Tissue distribution
|
Abstract
VNP40101M
(1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(2 methylamino)carbonyl] hydrazine), a
novel DNA alkylating agent, is currently under clinical development for the treatment
of cancer in Phase I clinical trials. This study investigated the pharmacokinetics,
mass balance, and tissue distribution of [
14
C]-VNP40101M in rats following a single intravenous dose of 10 mg/kg. After 7
days, the total recovery of radioactivity was 85% for males and 79% for females.
Most of the radioactivity was eliminated within 48 hours through urine (70%), with
less excreted in feces (6%). Tissue contained relatively high radioactive residues
with the highest concentrations in kidneys, liver, lung, and spleen. After 7 days,
tissue still contained 9% of the dose. At both 5 minutes and 1 hour post-dose, brain
contained relatively high radioactivity (5.9 and 3.3 µg equivalence/g and 50%
and 30% of the blood concentration, respectively), suggesting that VNP40101M
penetrated the blood-brain barrier. The elimination half-life of VNP40101M was
approximately 20 minutes, the peak plasma concentration (Cmax) averaged 11.3
µg/mL, the volume of distribution (Vss) averaged 0.91 L/kg, and the total body
clearance (Cl) averaged 33.5 mL/min/kg. The metabolite profile in urine was complex,
indicating VNP40101M was extensively metabolized. There were no apparent sex
differences in pharmacokinetic parameters of VNP40101M in the rat.
Introduction
DNA alkylating or cross-linking agents are among the most effective therapeutic
agents to treat different malignancies. These agents have shown broad activity
against human cancers and are a component of many standard treatment regimens in
current practice. The mechanism of action for all alkylating agents involves
damaging tumor DNA and impairing DNA replication in the tumor. Among this class of
agents, some display different spectrums of anti-tumor activity and toxicity. To some
degree, the differences in activity and toxicity can be accounted for by differences
in the type of DNA damage, the specificity for attacking DNA versus other cellular
components, the mechanisms by which the cell repairs the particular type of DNA
damage, and entry into and disposition of the drug within the tumor and normal
cells. Therefore, it becomes possible to create novel alkylating agents with the
potential for improved anti-tumor activity and a superior safety profile compared to
currently available agents.
VNP40101M belongs to a class of sulfonylhydrazine prodrugs that are potent
alkylating agents and have broad anti-tumor activity in animal models.
1-5
It is a relatively specific O
6
guanine chloroethylator, produces minimal or no alkylation of the N
7
position of guanine, and rarely induces single-strand DNA breaks, thus
maximizing DNA damage associated with anti-tumor activity and minimizing damage
associated with toxicity.
6
The metabolism of VNP40101M leads to a high yield of hard chloroethylating
species
6
and also yields a methyl-isocyanate that inhibits the cellular enzyme O
6
alkylguanine DNA alkyltransferase (AGT), which removes the initial
mono-adducts in DNA and prevents cross-linking. VNP40101M is active against mer+
(high AGT) cell lines
7
and is active against cell lines with high glutathione and glutathione
tranferase activity, which are known mechanisms of resistance to alkylating
agents.
8
Complete cures (survival >60 days) were found in L1210 carcinoma-bearing mice
administered with VNP40101M at doses of 10 and 15 mg/kg given daily for 6 days
following tumor implantation.
5
VNP40101M was also shown to cross the blood-brain barrier and eradicate
leukemia cells with a log kill of greater than 6.
9
In addition, VNP40101M has increased solubility compared to other agents in
this class,
10
although its solubility is modest. Because of these specific properties,
VNP40101M was selected for clinical development and is currently in Phase I safety
trials.
The purpose of this study was to assess the pharmacokinetics, mass balance, and
tissue distribution of VNP40101M in rats following a single IV radioactive dose. The
study was conducted in Sprague-Dawley rats at a nominal dose of 10 mg/kg. Plasma
samples were collected up to 8 hours post-dose and used for pharmacokinetic (PK)
analysis. For determination of excretion and mass balance, urine, feces, and cage
rinse were collected up to 7 days post-dose and analyzed for radioactivity. Groups
of animals were killed at 5 minutes, 1 hour, 24 hours, and 7 days post-dose to
determine radioactivity distribution in various tissues. Mass balance and tissue
distribution data as well as PK parameters of VNP40101M in rats are presented.
Materials and Methods
Materials
[
14
C]-VNP40101M (
Figure 1
) with a radiopurity of 99.9% and specific activity of 55 mCi/mmol was
purchased from Moravek Biochemicals (Brea, CA). Non-radiolabeled VNP40101M with
a chemical purity of 99.1% was manufactured at Vion Pharmaceuticals Inc (New
Haven, CT) and used as a reference standard. A solution formulation of VNP40101M
used to prepare the dose solution contained the following components: 100 mg
VNP40101M, 7 mL PEG-300, 3 mL ethyl alcohol 200 proof, dehydrated.
11
Blank vehicle was used to dose the control animals. Acetonitrile, formic
acid, isopropyl alcohol (IPA), and methanol were obtained from EM Science
(Gibbstown, NJ). Absolute alcohol was obtained from Quantum (Morris, IL).
Ammonium hydroxide, ammonium phosphate, and citric acid were obtained from J.T.
Baker (Phillipsburg, NJ). Heparin (sodium salt) was purchased from
Aldrich (Milwaukee, WI). Normal saline (0.9% NaCl) was obtained from
Baxter (Deerfield, IL). Water used in this study was produced in the
laboratory through a NANOPure® II (Barnstead Co, Dubuque, Iowa) water
purification system. Scintillation Cocktails were purchased from R. J. Harvey
Instrument Corporation, (Hillsdale, NJ) or Beckman Instruments,
Inc (Somerset, NJ). All other solvents and reagents were of
high-performance liquid chromatography (HPLC) grade and were purchased from
either Aldrich Chemical Co or Fisher Scientific Co (Pittsburgh, PA).
Animals
Adult male and female Sprague-Dawley rats (Hilltop Lab Animals, Inc,
Scottdale, PAB), 6-10 weeks old, body weights ranged from 234-258 g [male] and
227-246 g [female] at dosing) were used in the mass balance and tissue
distribution groups. Jugular vein-cannulated rats (Hilltop Lab Animals, Inc,
Sprague-Dawley, 6-10 weeks old, body weights ranged from 288-300 g [male], and
263-285 g [female] at dosing) were used in the PK group. All animals were
acclimated for at least 5 days before dosing. Care of the animals was in
accordance with institutional guidelines. Food and tap water were available ad
libitum. Dose Preparation
Dosing solution was prepared as follows: ~0.975 mCi of [
14
C]-VNP40101M (in acetonitrile) was transferred into a tared vial and the
solvent was evaporated. An aliquot of 12 mL of a liquid formulation of
VNP40101M (containing 10 mg VNP40101M/mL) and 24 mL 0.9% NaCl (Baxter USP) was
added to the vial and stirred for 10 minutes. Absolute ethanol (6 mL) was then
added and the solution was stirred and sonicated until a clear solution
resulted. The concentration of the dosing solution was assayed by HPLC in
duplicate before and after dosing and radiopurity confirmed. Study Design and Dosing Regimen
Male and female rats, housed individually in stainless steel metabolism
cages, were each given a single IV bolus dose of [
14
C]-VNP40101M at a nominal dose rate of 10 mg/kg body weight (the highest
nontoxic dose in rats). Three animals per gender were used in Group 1. Plasma was
obtained from these animals at pre-dose and at 2, 5, 15, 30, and 60 minutes and
2, 4, and 8 hours post-dose. Samples were analyzed for VNP40101M by HPLC.
Three animals per gender were used in Group 2. Urine was collected at 0-4 hours,
4-8 hours, and 8-24 hours, and then daily until 7 days post-dose. Feces samples
were collected daily post-dose for a total of 7 days. On day 7, animals were
killed for tissue distribution determination. During the study, cages were
rinsed daily with water and the cage rinse was collected. At the end of the
study, the cages were washed thoroughly with isopropanol/water (1:1). Sample
weights at each interval were determined at time of collection. Nine animals per
gender were used in Group 3. Three animals per gender were killed at 5 minutes
and at 1 hour and 24 hours post-dose for tissue distribution determination. One
animal per gender (Group 4) was injected with blank vehicle and animals were
killed at 24 hours post-dose. Urine, feces, and tissues were collected; no
radioanalysis was done on these samples. Sample Collection
For chemical specific analysis of plasma and urine by HPLC, samples were
acidified prior to analysis to stabilize VNP40101M since VNP40101M undergoes
hydrolytic degradation especially under basic conditions.
10,11
Acidification procedures were evaluated through a series of experiments
(results not shown). It was determined that a volume of 1M citric acid
equivalent to approximately 5% of the sample volume was needed to stabilize
VNP40101M in both blood and urine. Once acidified, VNP40101M is stable during
freezer storage and freeze/thaw. For total radioactivity determination (ie,
liquid scintillation counting [LSC]), acidification of samples was not carried
out.
Plasma: Blood was collected from Group 1 animals via jugular vein cannula. Prior
to collecting each PK sample, a small blood sample (approximately 0.05 mL) was
drawn and discarded. After each sample collection, saline (0.5 mL) was injected
to flush the catheter, and a heparin lock was then placed. An approximately
0.25-mL blood sample was collected into a syringe (using sodium heparin as
anticoagulant), transferred into microcentrifuge tubes, and placed on wet ice
immediately. All blood tubes were centrifuged at 10 000 rpm for 10 minutes at
4°C in a Beckman GS-15R centrifuge within 30 minutes after collection.
After centrifuging, 100 mmL of plasma was pipetted into another tube, 5 mmL of
1M citric acid was added into each plasma sample and mixed well. The plasma
samples were stored in a freezer (-20°C) prior to HPLC analysis.
Urine, Cage Rinse, and Feces (from Group 2 animals): Urine was collected
into containers surrounded by dry ice. Cages were rinsed with Nanopure®
water on a daily basis for 7 days post-dose. Feces were collected from a
stainless steel screen placed at the bottom of the cage and transferred to a
specimen cup on a daily basis for 7 days post-dose at room temperature. Upon
collection, feces were stored in a freezer until analysis. Sample weights were
recorded upon collection. Urine and cage rinse were aliquoted and analyzed for
radioactivity immediately.
Tissues: Animals from Groups 2, 3, and 4 were killed at the end of each
study. Group 3 animals were killed (3/time point/gender) at 5 minutes and 1 hour
and 24 hours post-dose. These time points were selected in an attempt to capture
peak tissue concentrations as well as to measure the rapid elimination of
VNP40101M. Animals were anesthetized by an overdose of CO
2
, and then ~5-7 mL of blood was collected into a heparinized Vacutainer by
cardiac puncture. Blood was placed on wet ice upon collection. Tissues from
liver, kidneys, spleen, muscle, bone, lungs, heart, brain, and carcass were
collected from each animal, rinsed with saline, and blotted dry on tissue paper.
Fat (abdominal) and skin (dorsal) were also collected from Group 2 animals.
The weight of each tissue was recorded. Sample Preparation and LSC Analysis
Upon collection, the weight of each urine sample was determined, and an
aliquot was acidified immediately with appropriate volume of 1M citric acid
equivalent to ~ 5% of the urine weight to bring the pH to ~4.5. Duplicate
aliquots of approximately 0.1 mL urine and 1 mL cage rinse were mixed with 5 mL
Ready Value Scintillation Cocktail (Beckman), and counted directly by liquid
scintillation counter. The acidified urine was kept frozen and used for HPLC
analysis. Feces were mixed with 5-10 volumes of water:acetonitrile (1:1). The
weight of the mixture was recorded and then homogenized for 2 minutes using a
Tekmar tissuemizer® (Cincinnati, OH). Triplicate aliquots of feces
homogenate (~0.6 g or less if the radioactivity level is expected to be high)
were combusted using a Harvey Biological Sample Oxidizer (Hillsdale, NJ).
Tissues (except bone, muscle, skin, and fat) were homogenized for 2 minutes with
2 volumes of water using a Tekmar tissuemizer. Duplicate aliquots (~0.3 g) were
air dried followed by combustion. Bone, muscle, skin, and fat were minced with
scissors, and approximately 0.1 g of bone, muscle, and skin and 0.05 g of fat
were combusted in duplicate. HPLC Analysis
A Agilent Technoligies 1100 liquid chromatography system (Wilmington, DE)
was used for the analysis of plasma and urine samples. The HPLC system consisted
of a quaternary pump, an autosampler, a column heater, and a β-RAM Model 2
flow-through radiometric detector (IN/US Systems,Tampa, FL). Plasma samples were
analyzed using a Phenominex Prodigy column (Torrance, CA) (5 µm, 250 Ă—
2.0 mm) at 40şC. The mobile phases were A) 0.1% acetic acid, and B)
acetonitrile. A linear gradient program was used: 0 minute, 90%A/10%B; 30
minutes, 50%A/50%B. The flow rate was 0.4 mL/min. A flow-through radiometric
detector equipped with a liquid scintillation cell (1 mL) was used to monitor
radioactive components. The scintillation cocktail was set at 1.2 mL/min. The
run time was 20 minutes with a 10-minute equilibration delay. For urine samples,
a Phenominex Prodigy column (5 µm, 250 Ă— 4.6 mm) was used, and the flow rate
was 1 mL/min. The gradient program was: 0 minute, 90%A/10%B; 30 minutes,
40%A/60%B. The scintillation cocktail flow was 3 mL/min. The run time was
20-30 minutes with a 6-minute equilibration delay.
Each plasma sample (0.05-0.1 mL) was extracted with 0.2 mL of methanol by
vortexing thoroughly. The extract was centrifuged at 15 000 rpm for 15 minutes at
room temperature. The supernatant was evaporated to dryness under a stream of
nitrogen at room temperature. The residue was reconstituted with 0.1 mL of a
dilution solvent consisting of acetonitrile and 0.1% acetic acid (1:9). The
pre-extraction sample and extract (5 µL each) were analyzed by LSC for total
radioactive residues. Each extract (30 µL) was analyzed by HPLC with
radiometric detection for VNP40101M and metabolite distribution. Each urine
sample (0.1 mL) was mixed thoroughly with 0.1 mL of acetonitrile. The extract was
centrifuged at 15 000 rpm for 15 minutes. The supernatant was transferred into an
HPLC vial and analyzed by HPLC with radiometric detection. The pre-extraction
sample and extract (10 µL each) were also analyzed by LSC for total
radioactive residues. HPLC calibration standards were prepared in blank rat
plasma and urine with [
14
C]-VNP40101M. The calibration range was 0.14-14.2 µg/mL for plasma,
and 2.2-141.5 µg/mL for urine. Calibration curves were constructed and
VNP40101M concentrations in plasma and urine samples were calculated based on
peak area. Pharmacokinetic Analysis
Pharmacokinetic parameters for VNP40101M in plasma were calculated with a
1-compartment model using WinNonlin software (Pharsight Corporation, Mountain
View, CA). An appropriate pharmacokinetic model was chosen based on the lowest
weighted squared residuals, lowest Schwartz Criterion (SC), lowest Akaike's
Information Criterion (AIC) value, lowest standard errors of the fitted
parameters, and dispersion of the residuals. The elimination half-life (T
1/2
) was estimated by linear regression analysis of the terminal phase of the
plasma concentration-time profile. The area under the concentration time curve
(AUC) was calculated between the first and last sampling times. Peak plasma
concentration (Cmax), total body clearance (Cl), and volume of distribution at
steady-state (Vss) were also calculated.
Results
Animal Health and General Observation
At a dose of 10 mg/kg of [
14
C]-VNP40101M, animals appeared healthy throughout the study. Male animals
gained a reasonable amount of body weight, females did not gain much weight, and
some even lost a little weight over the 7-day study period in Group 2. Excretion Pattern and Mass Balance
Urine, cage rinse, and feces from Group 2 animals were collected and
analyzed for total radioactivity. After 7 days, male rats recovered 65.6%, 4.9%,
and 5.5% of administered dose in urine, cage rinse, and feces, respectively,
while female rats recovered 61.0%, 3.7%, and 5.1% in urine, cage rinse, and
feces, respectively. Tissues and carcass contained 8.8% and 9.4% of administered
dose in male and female rats, respectively, on day 7. Overall recoveries were
84.9% in male and 79.2% in female rats. Most of the radioactivity (males
excreted 68.9% and females excreted 63.4% of the dosed radioactivity) was
excreted during the first 48 hours.
Table 1
summarizes the total radioactivity excreted from urine, cage rinse, and
feces over a period of 168 hours as well as residual radioactivity in tissues at
168 hours post-dose. The excretion pattern of [
14
C]-VNP40101M-derived radioactivity is shown in
Figure 2
. Approximately 15% and 21% of administered radioactivity was not accounted
for in male and female rats, respectively. The consistency of the data among all
animals and the
14
C-label position of VNP40101M suggests the loss of mass balance might be
due to the generation of radioactive CO
2
or other volatile species since a closed system was not used in this
study.
Tissue Distribution
Selected tissues (brain, heart, kidneys, liver, lungs, muscle, blood,
spleen, bone, fat, and skin) were collected and analyzed for total radioactivity
concentration in Group 3 animals at 5 minutes and at 1 hour and 24 hours
post-dose, and in Group 2 animals at 168 hours post-dose. Tissue radioactivity
distribution data are presented in
Tables 2
and
3
. At 5 minutes post-dose, all of the tissue analyzed contained a
significant amount of radioactivity, with the highest concentration in kidneys
(24.1 mmg equivalence/g in males and 14.5 mmg equivalence/g in females), liver
(17.7 mmg equivalence/g in males and 14.9 mmg equivalence/g in females), and
lungs (14.0 mmg equivalence/g in males and 14.9 mmg equivalence/g in females).
Muscle contained the highest percentage of radioactivity (44.0% in males and
48.9% in females), followed by blood (8.2% in males and 8.7% in females) and
liver (7.9% in males and 5.4% in females). At 1-hour post-dose, most tissue
concentrations decreased to ~48%-80% of the 5-minute level, except kidneys and
blood. At 24-hours post-dose, blood contained the highest concentration of
radioactivity (5.9 mmg equivalence/g in males and 7.1 mmg equivalence/g in
females). Approximately 11%-12% of administered radioactivity still remained in
the tissue at this time point. At 168 hours post-dose in male rats, only blood
contained significant radioactivity (3.5 mmg equivalence/g). In female rats,
blood radioactivity concentration was 4.5 mmg equivalence/g, whereas heart,
lungs, and spleen contained 1.2, 1.5, and 1.4 mmg equivalence/g, respectively.
The percent of dosed radioactivity in blood, muscle, bone, skin, and fat was
normalized to the total body mass of each tissue.
12,13
Therefore, for 168-hour tissue recovery, the total percent of dose should
represent a majority of the total tissue residue. It should be noted that brain
contained a significant concentration of
14
C residue (5.9 mmg equivalence/g at 5 minutes post-dose, 2.8-3.8 mmg
equivalence/g at 1 hour, and 0.7-0.9 mmg equivalence/g at 24 hours), indicating
that VNP40101M passed the blood-brain barrier.
RadioHPLC Analysis of Plasma and Urine
Calibration standards of [
14
C]-VNP40101M were prepared in blank rat plasma and urine and used to
evaluate the analytical method. Based on total radioactivity by liquid
scintillation counting, the extraction efficiency averaged 89% ± 18% (n=16)
and 106% ± 12% (n=10) for plasma and urine standards, respectively. By
radioHPLC, the percent recovery of the back-calculated concentration of VNP40101M
averaged 101% ± 8% in plasma and 100% ± 12% in urine. Calibration
curves were linear with r
2
values greater than or equal to 0.9875 in both matrices. Under the HPLC
conditions, VNP40101M eluted at approximately 13.5 minutes in the plasma method
and 16 minutes in the urine method. The acidified plasma and urine samples from
the study were extracted and analyzed for quantitation of VNP40101M and
metabolite profiles. At least 4 polar (eluted before parent drug with a relative
retention time <0.4) metabolites were observed in plasma samples, and their
quantities increased with time. In urine, the metabolites were predominant and
the chromatographic profile was complex. At least 6 very polar metabolites were
observed. Among the radioactive residues excreted in the urine over the 7-day
period (71% in males and 65% in females), more than 85% were metabolites
indicating that VNP40101M was extensively metabolized. The low renal clearance
of VNP40101M suggests that renal excretion was not a major route of elimination
of VNP40101M. Pharmacokinetics in Plasma
Pertinent pharmacokinetic parameters of VNP40101M in rats are shown in
Table 4
. The elimination of VNP40101M in rats following IV administration was
rapid with a mean elimination half-life of approximately 20 minutes. Plasma
VNP40101M concentration was the highest at 2 minutes post-dose with a mean peak
concentration of approximately 11 µg/mL. By 2 hours, VNP40101M was
generally below the method quantitation limit (<0.28 µg/mL). The plasma
concentration time profile is shown in
Figure 3
. The mean total body clearance of VNP40101M (33 mL/min/kg) was high
relative to hepatic (55 mL/min/kg) and renal (37 mL/min/kg) blood flow rates of
the rat.
14
The mean volume of distribution (0.9 L/kg) of VNP40101M approximated the
total body water (0.67 L/kg) in the rat.
14
It should be noted that the total body clearance for males
(41.7 mL/min/kg) is quite different from that of females (25.3 mL/min/kg). Due
to the limited number of animals, it is not clear at this time if this difference
is real or within experimental variability.
Discussion
Following an IV dose of 10 mg/kg in the rat, [
14
C]-VNP40101M was eliminated rapidly with a terminal half-life of
approximately 20 minutes. Radioactivity was extensively distributed into tissues
of rats. Among the tissues, the kidneys, lung, liver, and blood contained the
highest radioactivity on a µg/g of tissue basis. Elimination of
radioactivity from the tissues was prolonged with approximately 9% of the dose
still remaining after 7 days. A total of approximately 82% of the administered
radioactivity was recovered after 7 days. The remaining 18% was presumably due
to the formation of volatile metabolites (ie,
14
CO2), which were not trapped in this study. Of the excreted radioactivity,
approximately 70% was found in urine, and 6% in feces. The excretion was rapid
with >60% of the administered radioactivity (80% of total excreted) excreted
within 48 hours. Brain was found to contain relatively high radioactive
residues (50% and 30% of the blood concentration at 5 minutes and 1 hour,
respectively), indicating that VNP40101M penetrated the blood-brain barrier. The
curves for total radioactivity and VNP40101M concentration in plasma (
Figure 3
) diverged quickly, suggesting radioactive metabolites were formed at a
very early stage. This implies that the radioactivity found in tissues does not
represent VNP40101M, even as early as 1 hour.
The metabolism of [
14
C]-VNP40101M was also investigated in this study although identification of
metabolites was not attempted. The rapid elimination of VNP40101M in plasma and
the formation of complex polar metabolites were consistent with the expected in
vivo metabolic fate of VNP40101M.
5,9
A known degradation product of VNP40101M,
1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine
9,10
was not detected in either plasma or urine samples. The absence of this
compound in plasma and urine at study sampling intervals was consistent with its
reactive and transient nature.
9,10
In conclusion, the pharmacokinetics of VNP40101M in rats primarily involves
metabolism (and/or hydrolytic degradation) of VNP40101M followed by urinary
excretion of its polar metabolites. The wide spread of radioactivity in various
tissues and the relatively slow tissue clearance of VNP40101M metabolites are
consistent with the DNA alkylating properties of VNP40101M. The findings in this
study provide the basis for understanding the distribution, metabolism, and
elimination characteristics of VNP40101M in cancer patients.
Acknowledgements
We thank Dr Alan Sartorelli, Dr Terry Doyle, and Dr Mario Sznol for providing
background information on VNP40101M and for their general support in the research of
VNP40101M. We also like to thank Dr Caroline Clairmont and Paula Lambert for their
assistance during the study conduct and manuscript preparation.
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