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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
JPET 282:1173–1180, 1997
Vol. 282, No. 3
Printed in U.S.A.
Phase I Safety and Pharmacokinetic Profile of an Intercellular
Adhesion Molecule-1 Antisense Oligodeoxynucleotide (ISIS
2302)1
JOSEPHINE M. GLOVER, JANET M. LEEDS, TIMOTHY G. K. MANT, DIPTI AMIN, DANIEL L. KISNER,
JOAN E. ZUCKERMAN, RICHARD S. GEARY, ARTHUR A. LEVIN and WILLIAM R. SHANAHAN, JR.
Isis Pharmaceuticals Inc., Carlsbad, California (J.M.G., J.M.L., D.L.K., J.E.Z., R.S.G., A.A.L., W.R.S.) and Guy’s Drug Research Unit Ltd,
London SE1 1YR, United Kingdom (T.G.K.M., D.A.)
Accepted for publication May 12, 1997
Multiple cell surface molecules responsible for activation
and suppression of the immune system have been described
and characterized (Springer, 1990). One such molecule,
ICAM-1, is a transmembrane glycoprotein constitutively expressed at low levels on vascular endothelial cells and on a
subset of leukocytes (Dustin et al., 1986; Rothlein et al., 1986;
Simmons et al., 1988). In response to pro-inflammatory mediators, many cell types will up-regulate expression of
ICAM-1 on their surface. ICAM-1 binds to the b2 integrins,
LFA-1 and Mac-1, expressed on leukocytes (Marlin and
Springer, 1987; Diamond et al., 1990) and serves multiple
functions in the propagation of an inflammatory process, the
best characterized being facilitation of leukocyte emigration
in response to inflammatory stimuli (Butcher, 1991; Furie et
al., 1991; Oppenheimer-Marks et al., 1991).
Numerous studies have demonstrated an increase in
ICAM-1 expression within tissues obtained from patients
suffering from diseases with an inflammatory component.
Although it is unlikely that increased ICAM-1 expression is
Received for publication October 29, 1996.
1
This study was funded by Isis Pharmaceuticals, Inc.
(53–54 min) and total oligonucleotide (67–74 min) were similar
at the two doses (0.5 and 2.0 mg/kg) at which extensive pharmacokinetic data were collected. Nonlinear changes in area
under the plasma concentration/time curve and steady-state
volume of distribution with increasing dose suggested a saturable component to disposition. Metabolites co-migrating with
n-1, n-2 and n-3 chain-shortened versions of ISIS 2302 appeared very rapidly in plasma, and disposition and metabolism
appeared unaltered by repeated dosing. ISIS 2302 was well
tolerated and behaved reproducibly with respect to plasma
pharmacokinetics and expected side effects.
the causative event for these diseases, continued expression
of ICAM-1 probably contributes to the pathophysiology.
ICAM-1 monoclonal antibodies have been used to demonstrate beneficial effects in a variety of animal models of
disease, including pulmonary inflammation and asthma
(Barton et al., 1989; Wegner et al., 1990), prevention of allograft rejection (Cosimi et al., 1990; Isobe et al., 1992), nephritis (Harning et al., 1992; Kawasaki et al., 1993), ischemic
injury (Ma et al., 1992; Kelly et al., 1994), arthritis (Iigo et al.,
1991) and contact dermatitis (Scheynius et al., 1993), which
lend further support to the hypothesis that inhibitors of
ICAM-1 function or expression could have broad therapeutic
benefit.
ISIS 2302 is a phosphorothioate oligodeoxynucleotide 20
bases in length, designed to hybridize to the 39-untranslated
region of human ICAM-1 mRNA. ISIS 2302 selectively inhibits cytokine-induced ICAM-1 expression on a wide variety of
human cells in vitro (Bennett et al., 1994; Mielo et al., 1994;
Nestle et al., 1994). A murine equivalent, ISIS 3082, has been
shown to be active in multiple murine pharmacology models
of inflammation including prolongation of cardiac allograft
survival (Stepkowski et al., 1994), dextran sulfate-induced
ABBREVIATIONS: ICAM-1, intercellular adhesion molecule-1; APTT, activated partial thromboplastin time; TT, thrombin time; PT, prothrombin
time; CGE, capillary gel electrophoresis.
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ABSTRACT
Healthy male volunteers received single or multiple intravenous
infusions of an intercellular adhesion molecule-1 antisense
phosphorothioate oligodeoxynucleotide, ISIS 2302, in a risingdose (0.06 –2.00 mg/kg infused over 2 hr), double-blind, placebo-controlled trial. Brief, dose-related increases in activated
partial thromboplastin time were seen at the time of peak
plasma concentration (Cmax). Clinically insignificant increases
in C3a were seen after higher, repeated doses, but C5a, blood
pressure and pulse were unaffected. No adverse events or
other laboratory abnormalities were related to treatment with
the drug. ISIS 2302 Cmax was linearly related to dose and
occurred at the end of infusion. Plasma half-life for intact drug
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Glover et al.
Vol. 282
colitis (Bennett et al., 1997) and endotoxin-induced pneumonitis (Kumasaka et al., 1996). In each study, control oligonucleotides failed to demonstrate pharmacological activity,
which suggests that the anti-inflammatory activity of ISIS
3082 was caused by inhibition of ICAM-1 expression. Downregulation of ICAM-1 protein or message was demonstrated
in involved tissue in the colitis and pneumonitis models.
The purpose of this first clinical trial with ISIS 2302 was to
assess the safety and pharmacokinetics of intravenous administration of an anti-ICAM-1 oligodeoxynucleotide in
healthy subjects before commencing pilot therapeutic trials
in target disease states.
Methods
Results
Forty-four Caucasian, male subjects entered the trial,
ranging in age from 20 to 35 years. One subject failed to
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Study drug. ISIS 2302, a 20-base phosphorothioate oligodeoxynucleotide, was supplied by Isis Pharmaceuticals, Inc. as a sterile
solution of 2.5 mg/ml in phosphate-buffered saline (pH 7.6). The
percentage (area-percent) of full-length oligonucleotide, determined
by capillary gel electrophoresis, was 92.25%, with the major impurity
consisting of 4.95% n-1 deletion sequences. The product was 93.2%
fully thioated, with the occurrence of more than one nonthioated
(phosphodiester) linkage in a single molecule being rare. ISIS 2302 is
a racemic mixture, with an opportunity for chirality at each of its 19
phosphorothioate linkages.
Trial drug (or placebo) was administered by intravenous infusion
in a volume of 80 ml over 2 hr in all cases. ISIS 2302 was diluted, as
necessary, in sterile normal saline by a pharmacist at the trial unit.
Sterile normal saline was used as placebo.
Subjects and sampling schedule. This was a double-blind,
placebo-controlled study, conducted at Guy’s Drug Research Unit
Ltd. with local ethics committee approval. Four healthy male volunteers were recruited to each of seven single-dose groups (dose levels
0.06, 0.12, 0.24, 0.5, 1.0, 1.5 and 2.0 mg/kg ISIS 2302) and each of
four multiple-dose groups (dose levels 0.2, 0.5, 1.0 and 2.0 mg/kg ISIS
2302) after screening medical history, examination and laboratory
tests had shown no clinically significant abnormalities. One subject
in each dose group was allocated (by use of a randomization list) to
receive placebo while the others received active drug solution. Single-dose groups were dosed on day 1 only and multiple-dose groups
were dosed on days 1, 3, 5 and 7. Groups were studied in a rising-dose
fashion and multiple dosing commenced after the first five singledose groups had completed the trial.
Subjects remained recumbent, with continuous electrocardiogram
monitoring for 4 hr after the beginning of each infusion. The following were measured before and at intervals after each infusion: supine
blood pressure and pulse, clotting screen (APTT, TT, PT), serum
complement split products (C3a, C5a), neutrophil count (single-dose
groups only), urine microproteins (retinol binding protein, N-acetylglucosaminidase, microalbumin; single-dose groups only) and standard laboratory safety screen (hematology, blood biochemistry, urinalysis). Serum samples were collected from multiple-dose groups at
14 and 21 days after the last infusion to be analyzed for the presence
of antibodies to ISIS 2302.
Blood samples were taken for plasma ISIS 2302 concentration
before and up to 24 hr after the beginning of infusion from all dose
groups. More complete pharmacokinetic profiling was performed and
urine collections were made up to 12 hr after the beginning of
infusion in 0.5 and 2.0 mg/kg single- and multiple-dose groups.
Safety analyses. Laboratory safety screens, neutrophil counts,
clotting screens and urine microproteins were analyzed according to
standard, validated, laboratory procedures. Complement split products were measured by the Children’s Nationwide Kidney Research
Laboratory, Guy’s Hospital, London, UK, with use of commercially
available C3a and C5a des Arg 125I assay kits (Amersham Life
Science, Arlington Heights, IL).
Procedure for detection of antibodies to ISIS 2302. This
procedure was performed by Isis Pharmaceuticals Inc, Carlsbad, CA,
with a modification of a previously described methodology (Lacy and
Voss, 1989). Disposable enzyme-linked immunosorbent assay plates
(Corning Costar, Oneonta, NY) were coated with ISIS 2302. Uncoated areas on plates were blocked by incubating with 2% bovine
serum albumin solution (Sigma Chemical, St Louis, MO) or 2%
non-fat-dried Carnation milk powder. Aliquots of the diluted plasma
samples (1:10 or 1:100) or equivalent volumes of diluted medium
from a hybridoma cell culture line producing monoclonal antibodies
which recognize ISIS 2302 (positive control) were added to the plates
in triplicate, then incubated. Because isolated ISIS 2302 (and other
phosphorothioate oligonucleotides) do not appear to be antigenic,
these monoclonal antibodies to ISIS 2302 were raised by immunizing
mice with ISIS 2302 conjugated to keyhole limpet hemocyanin.
Plates were washed and then incubated with goat anti-human IgG or
IgM conjugated with alkaline phosphatase (Sigma Chemical, St
Louis, MO). After four further washes, plates were incubated with
the alkaline phosphatase substrate, p-nitrophenylphosphate (Sigma
Chemical, St Louis, MO) and the absorption at 405 nm was determined spectrophotometrically with a Titertek Multiskan MCC/340
plate reader (Lab Systems, Helsinki, Finland). Wells incubated with
only a blocking agent (2% bovine serum albumin solution or 2%
non-fat-dried milk) served as negative controls for these samples.
Procedures for analysis of ISIS 2302 and certain metabolites in blood and urine. Drug analysis was performed by CGE, as
described previously (Leeds et al., 1996), by Isis Pharmaceuticals,
Inc, Carlsbad, CA, on triplicate aliquots from each sample of plasma
and urine. Samples were prepared for CGE by strong anion-exchange solid-phase extraction followed by two desalting steps: elution from a reverse-phase solid-phase extraction column, and then
membrane dialysis. A phosphorothioate oligonucleotide composed of
27 thymidine nucleotides (T27) was added to both plasma and urine
as an internal standard. CGE was performed with a Beckman P/ACE
Model 5010 instrument (Beckman, Fullerton, CA) with a 27-cm
column. Oligonucleotides eluting from the column were detected by
ultraviolet absorption at a wavelength of 260 nm. The linear range of
concentrations of oligonucleotides detectable in plasma by this
method is 10 nM to 20 mM.
Concentrations of intact ISIS 2302 (full-length oligomer) were
calculated by determining the corrected area (area under the UV
absorbance curve divided by migration time) of the appropriate electropherogram peak, dividing by the corrected area of the internal
standard peak and multiplying by the known starting concentration
of the internal standard added. An additional correction for differences in the extinction coefficient between the T27 internal standard
(230 mM21) and ISIS 2302 (187.8 mM21) was made according to
Beer’s Law: A1/A2 5 (extinction coefficient1 3 path length1 3 concentration1)/(extinction coefficient2 3 path length2 3 concentration2). Concentrations of major metabolites (n-1, n-2 and n-3 shortmers) were calculated as above by use of the extinction coefficients
for standard oligonucleotides shortened from the 39 end.
Pharmacokinetic analysis. Mean plasma concentrations were
calculated for each sample and then for each dose group at each
protocol-specified time point. Computer fitting of intravenous infusion data from each subject in the 0.5 and 2.0 mg/kg dose groups was
performed by PCNONLIN 4.0 (Statistical Consultants, Inc., 1986).
Both one- and two-compartment models were evaluated and the data
were best fit to a one-compartment model without weighting, with
the Nelder-Mead Simplex algorithm to optimize parameter estimates. Differences in pharmacokinetic parameters between the 0.5
mg/kg and 2.0 mg/kg dose groups were tested by unpaired, twotailed, Student’s t test.
1997
Fig. 1. Median APTT (expressed as percent of control) after each of
four doses of ISIS 2302 (n 5 3) or placebo (n 5 4). Placebo or ISIS 2302
(0.2, 0.5, 1.0 and 2.0 mg/kg) was administered by 2-hr intravenous
infusion on days 1, 3, 5 and 7.
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was seen after the fourth dose for 2.0 mg/kg (fig. 3). The
magnitude of increase was not of clinical significance in any
individual. The maximum change in this parameter (up to
130% median increase from base line) was seen at 60 or 120
min after the beginning of infusion. Substantial recovery had
occurred by 4 hr, but sampling was not continued beyond this
point.
Electrocardiogram, supine blood pressure and pulse were
unaffected by dosing. No serious adverse events were reported nor was any particular adverse event seen occurring
with disproportionate frequency after active drug with the
exception of headache (seven ISIS 2302, one placebo; expected ratio 3:1), whose incidence was not related to dose of
ISIS 2302. Review of the median percent change from base
line in laboratory safety variables revealed no dose-related
differences between active drug- and placebo-treated subjects, with the exceptions of the above-mentioned changes in
APTT and C3a levels. Importantly, no consistent changes
were seen in white cell indices (including neutrophil count)
as a result of dosing with this ICAM-1 inhibitor.
Predose and day 22 plasma samples from each multipledose group subject were analyzed for IgG and IgM antibodies
to ISIS 2302. No indication was seen of the presence of
drug-specific antibodies in subjects treated with ISIS 2302
under the conditions of this trial.
Pharmacokinetics. Results are expressed as both the
amount of parent compound and of total oligonucleotide
present (defined as the sum of intact ISIS 2302 plus apparent
n-1, n-2 and n-3 chain-shortened metabolites). In plasma,
metabolites shorter than apparent 17-mer were not generally
detected (fig. 4). Although substantial amounts of material
co-migrated with shorter oligonucleotide standards in urine
(fig. 5), these were not quantitated because base-line separation was not achieved reliably.
Plasma concentration/time curves for subjects who received single infusions of 0.5 and 2.0 mg/kg are shown in
figure 6. Pharmacokinetic parameters were calculated for
each subject in the 0.5 and 2.0 mg/kg single-dose groups
(table 1) by use of an open, one-compartmental model, which
yielded a good fit to the data by both statistical and visual
criteria, although the plasma concentration/time curve appeared biphasic. Plasma half-life appeared consistent between doses. Although end-of-infusion plasma concentrations of ISIS 2302 increased linearly with increasing doses of
drug (fig. 7), AUC values increased disproportionately; a
4-fold increase in single dose produced a 7-fold increase in
AUC of both intact drug and total oligonucleotide. This suggestion of a saturable component to the disposition of ISIS
2302 is supported by the results of pharmacokinetic modeling, which demonstrated a reduction in volume of distribution and plasma clearance as dose increased (table 1), and by
observations in animals with other oligonucleotides (Srinivasan and Iversen, 1995; Rifai et al., 1996).
During 2-hr, single infusions of ISIS 2302, metabolites
co-migrating with synthesized n-1, n-2 and n-3 chain-shortened forms (shortmers) of the intact drug appeared very
rapidly in plasma, constituting 20% of total oligonucleotide
after 30 min of infusion. Interestingly, the relative proportion
of total oligonucleotide constituted by full-length oligo and
n-1, n-2 and n-3 shortmers remained relatively constant during the 2 hr of study drug infusion and for at least the 4 hr
postinfusion during which metabolites were measured.
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return for follow-up 7 days after dosing, but all other subjects
completed the trial procedures.
Safety. A consistent and dose-related increase in APTT,
whose magnitude varied between individuals but was of potential clinical importance in the highest dose groups, was
seen in subjects who received single or multiple doses of 0.5
mg/kg ISIS 2302 and above (fig. 1). The maximum increase in
APTT was seen between 1 and 2 hr after the beginning of
infusion, and values returned to base line (or below) within 2
to 4 hr after the end of infusion. The greatest increases in
APTT were seen in the 2.0 mg/kg single- and multiple-dose
groups. End of infusion (hour 2) APTTs ranged from 49.4 to
54.3 sec in the three 2.0 mg/kg single-dose subjects, and from
39.9 to 51.5 sec on days 1, 3, 5 and 7 in the three 2.0 mg/kg
multiple-dose subjects (normal range, 27.0–36.2 sec). Median
APTT values were 45.4, 46.9, 46.4 and 48.0 sec on days 1, 3,
5 and 7, respectively, in the multiple-dose 2.0 mg/kg subjects.
Comparison of results from single- and multiple-dose groups
showed no consistent tendency for exaggeration or attenuation of this effect after multiple dosing: the highest median
APTT values were seen on day 1 (first infusion) in the 0.5 and
the 1.0 mg/kg dose groups, and on day 7 (fourth infusion) in
the 2.0 mg/kg dose group. By combining the hour 2 APTT
values for all four infusion days, a linear relationship of
APTT to dose over the investigated multiple-dose range of 0.2
to 2.0 mg/kg can be appreciated (fig. 2).
Much smaller, clinically insignificant and less clearly doserelated increases in PT and TT occurred in subjects who
received ISIS 2302. TT rose slightly above the reference
range in some cases but PT did not. The maximum median
increases in these parameters (approximately 6% for PT and
10% for TT) occurred between 30 and 120 min after the
beginning of a single infusion, and recovery occurred within
2 hr from this time. Bleeding time was unaffected by the trial
drug.
Complement C5a split product concentrations remained
unchanged throughout the study. Single-dose groups showed
no difference in complement C3a concentrations between
subjects who had received active drug and placebo. Among
multiple-dose group subjects, a small increase in median C3a
concentration was seen after the fourth dose of 1.0 mg/kg
ISIS 2302 and the third dose of 2.0 mg/kg. A larger increase
Phase I Trial of ICAM-1 Antisense
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Glover et al.
Vol. 282
Fig. 2. APTT in seconds at hour 2 for the
multiple-dose groups (0.2, 0.5, 1.0 and 2.0
mg/kg). The data from the four doses (days
1, 3, 5 and 7) and the placebo controls have
been pooled. The dashed line connects the
mean values for each dose, and the solid
line is the ordinary least-squares regression line for the data (r2 5 0.56).
Therefore, intact drug remained the majority of oligonucleotide present until levels fell below the limits of detection (fig.
8).
There was no evidence of accumulation of oligonucleotide
in plasma with alternate day dosing by infusion over 2 hr:
comparison of end-of-infusion concentrations of intact drug
or total oligonucleotide after the first through the fourth
multiple dosings with 0.2, 0.5, 1.0 or 2.0 mg/kg ISIS 2302
failed to reveal a consistent trend to change in these values
(fig. 9). Furthermore, there were no clear differences in the
proportions or concentrations of intact drug and metabolites
over time between the first and fourth infusions, giving no
suggestion that repeated dosing with ISIS 2302 caused induction or inhibition of its own metabolism.
Urine samples from the 1.0 and 2.0 mg/kg multiple-dose
groups were analyzed for concentrations of intact drug and
metabolites. Very low concentrations of ISIS 2302 and metabolites were present in urine. Intact drug and the n-1, n-2
and n-3 shortmers could be measured, and the quantity of
shorter metabolites, although not well resolved, could be
Fig. 4. Representative electropherogram of ISIS 2302 and chainshortened metabolites extracted from plasma obtained from a subject
1 hr after the end of the 4th of four every-other-day 2-hr infusions of
ISIS 2302, 2 mg/kg. CGE provides resolution between intact ISIS 2302
(n) and shortmer metabolites (n-1, n-2, n-3, etc).
visually estimated from electropherograms. The amount of
intact drug excreted over 6 hr after the beginning of infusion
averaged approximately 0.05% of the administered dose, and
the estimated total excretion of parent drug and metabolites
in this period was less than 0.5% of the total dose.
Discussion
The first objective of this trial was to establish the safety of
a range of single and multiple doses of ISIS 2302, a drug
which had not previously been administered to humans. This
objective was completed successfully, allowing the drug to
progress into phase II trials in a variety of therapeutic indications.
In cynomolgus monkeys, ISIS 2302 has consistently caused
anticoagulant effects (increased APTT) and alternative pathway complement activation at plasma concentrations in excess of 30 and 50 mg/ml, respectively, effects which are re-
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Fig. 3. Median complement C3a split product concentrations after
each of four doses of ISIS 2302 (n 5 3) or placebo (n 5 4). The
horizontal line indicates the upper limit of the normal range. Placebo or
ISIS 2302 (0.2, 0.5, 1.0 and 2.0 mg/kg,) was administered by 2-hr
intravenous infusion on days 1, 3, 5 and 7.
1997
Fig. 6. Logarithmic mean plasma concentration/time profiles during
and after 2-hr infusions of 0.5 mg/kg and 2.0 mg/kg ISIS 2302.
lated in magnitude to peak plasma drug concentration
(Henry et al., 1997). In this trial, single- and multiple-dose
groups demonstrated increases in APTT at doses of 0.5 mg/kg
ISIS 2302 and above. These increases were very clearly doserelated in magnitude, just as peak plasma drug concentration was linearly related to dose, and did not appear to
attenuate or accumulate with multiple dosing. The peak anticoagulant effect was seen at the time of peak plasma concentration of both intact ISIS 2302 and total oligonucleotide
(at the end of infusion), and changes recovered rapidly and
spontaneously within 2 to 4 hr after the end of infusion, the
same period during which oligonucleotide was detectable in
plasma after higher doses. The mechanism of this effect is
not yet fully elucidated, but it is known that ISIS 2302, like
other phosphorothioate oligonucleotides, is a large, polyanionic molecule which binds reversibly to several proteins,
including thrombin (Henry et al., 1994). The reliable relationship between plasma drug concentration and anticoagulant effect will allow maximum doses/rates of infusion for
future trials to be calculated on the basis of maximum acceptable increase in APTT.
No effect on the C5a complement split product was seen,
but small, brief increases in C3a appeared after repeated
infusions of higher doses of ISIS 2302. The mechanism of
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complement activation or conversion by ISIS 2302 observed
in this study is also not known although this is also suspected
to be related to protein binding (Henry et al., in press, 1997)
or nonspecific enzymatic degradation by leukocyte proteases.
Nonclinical experiments are underway to investigate these
possibilities. Rapid fluctuations in neutrophil count and hemodynamic changes, thought to be related to complement
activation, have been observed at higher dose levels and
infusion rates with ISIS 2302 and other phosphorothioate
oligonucleotides in monkeys (Galbraith et al., 1994; Henry et
al., 1997). These variables were also measured at frequent
intervals after the beginning of infusion in the single-dose
subjects in this trial, but no significant changes nor any
trends to change were observed. It is likely that any phosphorothioate oligonucleotide of sufficient chain length will
cause similar effects on clotting and complement functions in
animals and humans if administered in such a way as to
exceed threshold peak plasma drug concentrations.
Clinically, ISIS 2302 was well tolerated. There were no
clinical signs, symptoms or changes in routine laboratory
safety parameters which were related to ISIS 2302. Specifically, there was no evidence of an effect of this ICAM-1
inhibitor on gross immune function or on the white blood cell
indices measured. This is not surprising because ICAM-1 is
only one of the many mediators involved in regulating overall
immune function. Consistent with experience at Isis in other
animal and clinical trials with similar oligonucleotides, there
was no evidence of antibody formation to ISIS 2302.
These results are also important in that this is the first
description of the use of CGE to measure nonradiolabeled,
systemically administered, phosphorothioate oligonucleotide
in humans. Other authors have described the human pharmacokinetics of intravenously injected 35S-labeled GEM 91 (a
25-mer) (Zhang et al., 1995), intradermal 14C-labeled afovirsen sodium (a 20-mer) (Crooke et al., 1994) and unlabeled
OL(1)p53 (a 20-mer) (Bayever et al., 1993), all phosphorothioate oligodeoxynucleotides.
The results of pharmacokinetic analysis in this trial are
generally consistent with those obtained with other systemically administered phosphorothioate oligonucleotides in animals (Cossum et al., 1993, 1994; Agrawal et al., 1995) and
humans (Bayever et al., 1993; Crooke et al., 1994; Agrawal et
al., 1995; Zhang et al., 1995), although there are differences
with respect to the importance of urinary excretion and the
presence of a terminal half-life. The present study demonstrates a rapid distribution phase, corresponding to the distribution phase seen in animal studies, and the volume of
distribution appears to be consistent with previous data. This
study differs from previous studies in that the terminal halflife described in studies with radiolabeled drug was not observed. This difference may be related to differences in assay
sensitivity and the ability to measure single-nucleotide metabolites by radiochemical analysis. However, compared with
unmodified oligonucleotides with phosphodiester backbones,
the plasma half-life of intact drug is long. An additional
difference between reports lies in the assessment of the importance of urinary excretion. In this study and those conducted with 14C-labeled afovirsen sodium (Cossum et al.,
1993, 1994; Crooke et al., 1994), urine was found to be a
minor route of excretion of oligonucleotides (or of radioactivity derived from drug), whereas studies with 35S-labeled 20and 25-mers showed substantial urinary excretion of drug-
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Fig. 5. Representative electropherogram of ISIS 2302 and shortmer
metabolites extracted from urine collected from 0 to 6 hr from a subject
during and after the 3rd of four every-other-day 2-hr infusions of ISIS
2302, 2 mg/kg. CGE provides resolution between intact ISIS 2302 (n)
and shortmer metabolites [n-1 (A), n-2 (B), n-3 (C), etc.].
Phase I Trial of ICAM-1 Antisense
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TABLE 1
Mean plasma pharmacokinetic parameters derived for ISIS 2302 and total oligonucleotide from those groups (n 5 3) which received a
single 2-hr infusion of 0.5 or 2.0 mg/kg ISIS 2302
0.5 mg/kg Dose Group
Parametera
V22 (ml/kg)
AUC (0–00) (mg/min/ml)
K10t1/2 (min)
Cmax (mg/ml)
Cmax/dose (mg/ml)
Clearance (ml/min/kg)
2.0 mg/kg Dose Group
ISIS 2302
Total
Oligonucleotide
ISIS 2302
Total
Oligonucleotide
155.4 6 13.6
249.7 6 43.8
54.4 6 16.1
1.60 6 0.16
3.21 6 0.33
2.07 6 0.48
121.9 6 5.6
392.7 6 92.9
66.7 6 18.2
2.30 6 0.26
4.60 6 0.52
1.33 6 0.34
97.5 6 7.1b
1824.6 6 111.1
52.9 6 6.0
10.3 6 0.06
5.17 6 0.34b
1.28 6 0.12b
77.5 6 5.3b
2765.0 6 30.0
74.1 6 7.7
15.5 6 1.10
7.75 6 0.65b
0.73 6 0.08b
a
Vss, steady-state volume of distribution; AUC(0 –`), area under the plasma concentration/time curve extrapolated to infinity; K10t1/2, plasma half-life; Cmax, maximum
plasma concentration.
b
P , .05, as compared with the 0.5 mg/kg dose group.
derived radioactive material, mostly in the form of metabolites (Agrawal et al., 1995; Zhang et al., 1995). These differences could be accounted for by a difference in the route of
excretion of carbon- and sulfur-related moieties resulting
from metabolism of phosphorothioate oligodeoxynucleotides.
Zhang et al. (1995) describe the urinary excretion of approximately 70% of GEM 91-derived 35S over 96 hr after a single
human dose, whereas Cossum et al. (1993, 1994) and Crooke
et al. (1994) found approximately 50% and 30% of afovirsen
sodium-derived 14C in expired air and only approximately
15% and 10% in urine after single rat and human doses,
respectively.
Bayever et al. (1993) used high-performance electrophoretic chromatography to measure cumulative urinary excretion of an unlabeled 20-mer phosphorothioate oligodeoxynucleotide (OL(1)p53) given by continuous intravenous
infusion at a rate of 0.05 mg/kg/hr (1.2 mg/kg/day) for 10 days
to five patients with acute myelogenous leukemia or myelodysplastic syndrome. Cumulative urinary excretion of apparently undifferentiated oligonucleotide ranged from 42 to 63%
of administered drug. Plasma drug levels were not measured
directly, but peak plasma concentrations (2.1– 6.4 mg/ml) and
half-life (4.9 –14.7 days) were calculated from the rate of
urinary excretion. There are considerable methodological differences between the high-performance electrophoretic chromatography technique used by Bayever et al. (1993) and the
CGE technique (Leeds et al., 1996) used in the present study
to measure drug concentrations, including methods of extraction, scale of analytic method and ability to identify intact
drug and metabolites. Furthermore, plasma half-life was not
directly measured by Bayever et al. (1993), but calculated
from urinary excretion. Differences in plasma half-life and
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Fig. 7. Plasma concentrations of ISIS 2302 determined at the end of
single, 2-hr infusions of ISIS 2302.
the relative importance of urinary excretion found in these
two studies could be caused by differences in dosing regimen
(continuous infusion for 10 days versus 2-hr infusions every
other day) and/or quantitative methodology. Continuous infusion might saturate plasma binding capacity, cellular uptake and/or renal proximal tubular reabsorption.
No other accounts of human pharmacokinetic results after
multiple dosing are available for comparison. In this trial, no
accumulation of drug or metabolites was seen in plasma, nor
was there any apparent change in the kinetics or metabolism
of ISIS 2302 in plasma with repeated administration. Similar
plasma pharmacokinetic behavior has been seen in monkeys
(Isis Pharmaceuticals; A. Levin, unpublished data). Although
tissue levels cannot be determined in clinical trials, we can
speculate, by extrapolation from animal data demonstrating
tissue half-lives on the order of 24 to 120 hr, depending upon
the tissue and the dosing regimen (Cossum et al., 1993; Isis
Pharmaceuticals, A. Levin, unpublished data), that concentrations of ISIS 2302 and its metabolites in target tissues
may be maintained with an alternate-day regimen.
This study showed that the peak plasma concentration and
AUC for total oligonucleotide was approximately 50% higher
than for ISIS 2302 alone. Nonclinical studies have indicated
that synthesized n-1, n-2 and n-3 chain-shortened metabolites are capable of inhibiting ICAM-1 expression and of
causing the toxicities typical of phosphorothioate oligonucleotides (Isis Pharmaceuticals, F. Bennett and A. Levin, unpublished data).
The observation that the relative proportion of total oligonucleotide constituted by full-length oligo and n-1, n-2 and
n-3 shortmers remained fairly constant throughout the 2-hr
infusion period and for at least 4 hr postinfusion suggests
that ISIS 2302 is shortened one base at a time by exonucleases that compete at a constant rate for substrate irrespective of chain length [at least $ n-3 mers (17-mers)] and base
sequence. This constancy of proportionality (i.e., the very
slow increase in the proportion of metabolites over time),
present by 30 min into the infusion, the time at which the
first pharmacokinetic samples were drawn, further suggests
that metabolism in plasma is largely complete within 30 min.
Potential explanations for this phenomenon include: 1) the
buildup in plasma of a nuclease-inhibitory metabolite; 2) the
presence of a significant impurity in the drug product with a
different rate of metabolism; and 3) a differential rate of
metabolism for the Rp and Sp diastereoisomers present at
each phosphorothioate linkage. The generation of an inhibi-
1997
Phase I Trial of ICAM-1 Antisense
1179
Fig. 9. Plasma concentrations of ISIS
2302 and metabolites at the end of infusion (hour 2) during multiple dosing. Bars
show concentrations of ISIS 2302 (n),
chain-shortened metabolites (n-1, n-2,
n-3) and total oligonucleotide (sum of n,
n-1, n-2 and n-3) on days 1, 3, 5 and 7.
Each bar represents the mean concentration from three subjects, with vertical
lines showing standard deviation.
tory metabolite seems implausible because the rate and pattern of metabolism appear constant through time and across
a 4-fold dose range. With the exception of chirality, the ISIS
2302 administered was high in analytical purity (see “Methods”), and therefore the presence of a significant impurity
seems an unlikely explanation. However, phosphorothioate
diastereoisomeric selectivity has been reported for exonucleases (Burgers and Eckstein, 1979; Spitzer and Eckstein,
1988), and this offers a potential explanation for the pattern
of metabolism observed in this study. If pharmacokinetic
modeling is performed assuming exonuclease base deletion in
sequence, a probability of 0.5 at each linkage for rapid (Sp) or
slow (Rp) cleavage and a “half-life” of 3 hr for the Rp isomers
and 3 min for the Sp isomers, a metabolic pattern very
similar to that observed in this study is produced. The absence of significant urinary excretion and the failure to detect
a buildup of metabolites, either long (17- to 19-mers) or short
(,17-mers), suggests that ISIS 2302 is principally cleared
from plasma by extravascular tissue distribution and subsequent cellular uptake.
The fact that the major plasma metabolites detected in this
study co-migrated with standards shortened by one, two or
three nucleotides from the 39 end of the ISIS 2302 molecule is
also consistent with a hypothesis that phosphorothioate oli-
gonucleotides undergo metabolism by exonucleases which
remove single bases in a sequential manner from the end of
the molecule. Alternatively, nuclease activity might remove
pairs or triplets of bases from the end of the molecule to
produce n-2 and n-3 metabolites. Although CGE cannot determine the sequence of the apparent 19-, 18- and 17-mers
seen, the former hypothesis is more consistent with the observed metabolic profile and is inviting in that phosphodiester oligodeoxynucleotides in plasma undergo exonuclease
digestion from their 39 end (Eder et al., 1991). We would
further hypothesize that the shortmers resulting from such a
process would eventually be catabolized in much the same
way as endogenous nucleotides. This hypothesis is supported
by the previous finding (described above) that approximately
50% of 14C radiolabel derived from a similar phosphorothioate oligonucleotide, labeled at the C-2 position of thymidine,
was eliminated from rats as carbon dioxide in expired air
(Cossum et al., 1993, 1994). Biliary excretion is unlikely to
play any significant role because fecal elimination of labeled,
intravenously administered phosphorothioates has been
minimal in rodents (Cossum et al., 1993; Agrawal et al.,
1995).
Overall, the predictability of the clinical profile and pharmacokinetics resulting from repeated infusions of ISIS 2302
Downloaded from jpet.aspetjournals.org at ASPET Journals on August 1, 2017
Fig. 8. Mean concentrations of intact ISIS 2302 and metabolites in plasma during and after 2-hr intravenous infusions of 0.5 or 2.0 mg/kg ISIS
2302.
1180
Glover et al.
in this study gave the confidence necessary to allow pilot
therapeutic trials in indications including renal transplantation, inflammatory bowel disease, rheumatoid arthritis and
psoriasis to begin.
Acknowledgments
The authors thank C.Frank Bennett, Ph.D. for his scientific review of this paper.
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