Aronex Pharmaceuticals, Inc., The Woodlands, Texas (T.L.W., S.A.B.,
N.C., P.A.C.), and
TSI Mason Laboratories, Inc., Cambridge,
Massachusetts (K.H., P.M.M., J.P.S.)
AR177 is a 17-mer oligonucleotide that has anti-human immunodeficiency
virus activity in vitro. The disposition of internally labeled 33P-AR177 was studied after the tail vein injection
of single and multiple doses (0.7 mg/kg) to rats. After a single dose,
the terminal half-life of AR177 in the blood and plasma was 367 and 271 hr, respectively, significantly longer than values reported for other oligonucleotides. Analysis of the AR177 tissue distribution showed that
the majority of the dose was distributed to the liver (40%), bone
marrow (17%) and renal cortex (15%) at 8 hr after single dosing.
Analysis of the AR177 concentrations in tissues showed that the highest
concentrations were achieved in the renal cortex (15.0 µg-eq/g),
liver (7.4 µg-eq/g), bone marrow (3.9 µg-eq/g), mesenteric lymph
node (3.0 µg-eq/g) and spleen (2.4 µg-eq/g) at 8 hr after single
dosing. The half-life in these tissues was 9.6, 7.7, 36.8, 10.0 and
30.8 days, respectively. Forty-eight hours after the last of seven i.v.
doses given every other day, the concentrations in tissues were as
follows: renal cortex, 39.9 µg-eq/g; liver, 33.9 µg-eq/g; bone
marrow, 12.7 µg-eq/g; spleen, 9.3 µg-eq/g; mesenteric lymph node,
5.1 µg-eq/g. Twenty-one days after administration of the last dose,
tissue concentrations were still high, as follows: renal cortex, 18.6 µg-eq/g; liver, 6.2 µg-eq/g; bone marrow, 12.5 µg-eq/g;
mesenteric lymph node, 3.9 µg-eq/g; spleen, 8.1 µg-eq/g. There was
low urinary and fecal excretion (urinary excretion of 12.8% and fecal
excretion of 6.0% of the total dose over 21 days) after a single dose.
Gel filtration and anion-exchange high-performance liquid
chromatography and electrophoretic analysis of the radioactivity in
tissues indicated that >90% of the radioactivity represented intact
AR177 for at least 7 days after drug dosing. These results demonstrate
that AR177 has an extended plasma, blood and tissue half-life, is
widely distributed and achieves high concentrations in lymphoid and
nonlymphoid tissues in rats.
 |
Introduction |
There are currently a number of
drugs approved for the treatment of HIV infection that are either
reverse transcriptase or protease inhibitors. To date, monotherapy with
any drug has resulted in ultimate therapeutic failure, at least in part
because of the development of resistance. Progression of the disease
continues to occur in treated patients, with decreases in
CD4+ T lymphocyte concentrations and the onset of
opportunistic infections leading to death. There is a consensus among
clinical investigators that combinations of drugs from multiple
therapeutic classes are required to provide effective treatment of HIV
infection (Schnittman and Fauci, 1994
; Lange, 1995). Indeed, many
studies have shown that combinations of drugs from each therapeutic
class provide greater effectiveness than each of the drugs given alone.
The utility of this approach is dependent upon the development of anti-HIV drugs whose mechanism of action involves targets other than
reverse transcriptase or protease. Among other possible molecular targets is HIV integrase, the enzyme responsible for catalyzing the
incorporation of viral DNA into human DNA.
AR177 (T30177; Zintevir) is an oligonucleotide with the sequence
5
-GsTGGTGGGTGGGTGGGsT-3, where s represents phosphorothioate linkages. AR177 is the most potent inhibitor of integrase described to
date, with an IC50 in the 30 to 80 nM range (Ojwang
et al., 1995; Mazumder et al., 1996). Previous
studies have shown that AR177 has antiviral activity against both
laboratory and clinical strains of HIV-1 in human lymphocytes and
macrophages (Ojwang et al., 1995). AR177 is relatively
resistant to serum nucleases (Bishop et al., 1996), is
devoid of the cardiovascular toxicity (Wallace et al.,
1996a) seen with total phosphorothioate oligonucleotides in monkeys
(Cornish et al., 1993) and does not cause any tissue damage
after multiple i.v. doses to monkeys (Wallace et al.,
1996b). AR177 is the first integrase inhibitor to enter human testing and is currently in single- and multiple-dose phase I clinical trials.
As part of the preclinical assessment of AR177, the pharmacokinetics and tissue distribution were determined for 21 days after single- and
multiple-dose administration of 33P-AR177 to rats. This is
the first published report of the pharmacokinetics and distribution of
an oligonucleotide after multiple doses.
| |
Materials and Methods |
Materials.
Nonradiolabeled AR177 was synthesized on a
Milligen 8800 oligonucleotide synthesizer and made into a stock
solution at 25 mg/ml in sterile phosphate-buffered saline. AR177 has a
molecular weight of 5793 daltons and is a fully neutralized sodium
salt. The structure of AR177 was characterized by phosphorus and proton nuclear magnetic resonance spectroscopy, sequencing, base composition, laser desorption mass spectrometry, anion-exchange HPLC and
polyacrylamide gel electrophoresis. The AR177 preparation was 94%
pure, according to HPLC and electrophoretic analysis. All analyses were
consistent with the proposed structure.
For HPLC analysis of plasma AR177, Tris was obtained from Fisher, NaBr
and NaCl were obtained from Sigma and methanol was purchased from J. T. Baker. The Gen-Pak Fax anion-exchange HPLC column (4.6 × 100 mm;
catalogue no. 15490) was purchased from Waters.
Preparation of 33P-AR177 dosing
solution.
AR177 was internally labeled with 33P
according to the method of Bishop et al. (1996)
(5-GTGGTGGGT*GGGTGGGT-3, where the asterisk indicates the position of
labeling). The radiochemical purity was 98% for the single-dose study
and 96% for the multiple-dose study. The dosing solution was composed
of unlabeled AR177 and 33P-labeled AR177 formulated in
phosphate-buffered saline to a final concentration of 1 mg/ml.
Single-dose study.
The pharmacokinetics and tissue
distribution of 33P-AR177 were studied in male
Sprague-Dawley rats after a single i.v. dose of 0.7 mg/kg. The rats
were dosed at 0.7 ml/kg using a 1 mg/ml solution. The single-dose study
was performed in two parts, with male rats that were 8 to 10 weeks of
age. In the first single-dose study, rats were used for collection of
tissues and body fluids up to 4 days after the single dose. These rats
(n = 35) had a body weight of 300 ± 12 g
(mean ± S.D.) and received a dose of 0.7 mg/kg with a specific
activity of 119 µCi/mg. The dose of radioactivity that these rats
received was 25.0 ± 2.4 µCi/rat (mean ± S.D.). The
single-dose study was extended when it was discovered that the drug had
a long half-life after a single dose. In the extended study, male rats
were used for collection of tissues and body fluids at days 7, 14 and
21 after a single dose. These rats (n = 9) had a body
weight of 255 ± 12 g (mean ± S.D.) and received a dose
of 0.7 mg/kg with a specific activity of 101 µCi/mg. The dose of
radioactivity that these rats received was 18.1 ± 3.7 µCi/rat
(mean ± S.D.). The mean dose of radioactivity for the single-dose
study (n = 44 rats) was 24 µCi/rat.
Radioactivity was determined in blood and plasma at 0.25, 0.5, 1, 1.5, 2, 3, 4, 8, 12, 18, 24 and 32 hr and 2, 3, 4, 7, 11, 14, 18 and 21 days
after the administration of 33P-AR177. The blood and plasma
pharmacokinetic data were derived from six rats at 0.25, 0.5, 1, 2 and
8 hr after dosing, five rats at 4 days and three rats at 1.5, 3, 4, 12 and 18 hr and 1, 1.33, 2, 3, 7, 11, 14, 18 and 21 days after dosing.
The blood was collected in EDTA, and the plasma was obtained by
low-speed centrifugation of the blood. Aliquots of the blood and plasma
samples were reserved for analysis of the radioactivity by HPLC and gel
electrophoresis. Blood and plasma were taken from each rat, but a
different set of rats was used at each time point. This was done to
minimize the number of rats that were used in the study and the amount of blood that was taken from each rat. Radioactivity was determined in
tissues at 1, 2, 4, 8, 24, 32 and 48 hr and 4, 7, 14 and 21 days after
the administration of 33P-AR177. These tissues were
adrenal, bone marrow, brain, eyes, liver, lungs, renal cortex, renal
medulla, mesenteric lymph node, skeletal muscle, skin, spleen, testes
and thymus. The tissue pharmacokinetic data were taken from three rats
at 1 hr, two rats at 2, 4, 8, 24 and 32 hr and 2, 7, 14 and 21 days and
five rats at 4 days. Total radioactivity in tissues was determined in
these rats. One additional rat was used to determine the presence of
intact 33P-AR177 in tissues by HPLC and/or gel
electrophoresis. Radioactivity was determined in cumulative urine and
feces samples at 4, 8 and 24 hr and 3, 4, 5, 6, 7, 10, 11, 12, 13, 14, 17, 18, 19, 20 and 21 days after the administration of
33P-AR177. The urinary and feces pharmacokinetic data were
derived from five rats from 4 hr to 4 days of collection and from three rats from 5 days to 21 days of collection. The blood, plasma, urine,
feces and tissues were stored frozen until analyzed.
Multiple-dose study.
The pharmacokinetics and tissue
distribution of 33P-AR177 were studied in male
Sprague-Dawley rats after up to seven i.v. doses given every other day
at 0.7 mg/kg (specific activity, 20 µCi/mg; 3.8 µCi/rat), using a 1 mg/ml solution. The rats (male) had a body weight of 273 ± 21 g (mean ± S.D.) and were 8 to 9 weeks of age. The body
fluids and tissues that were sampled were the same ones described for
the single-dose study. Blood and plasma pharmacokinetic data were
derived from three rats at 0.25, 1, 2, 4, 8 and 24 hr after dose 1, just before doses 2 to 7 and at 0.25, 0.5, 1, 2, 4, 6 and 8 hr and 1, 2, 3, 7, 14 and 21 days after dose 7. Aliquots of the blood and plasma
samples were reserved for analysis by HPLC and gel electrophoresis.
Tissue samples were collected from two rats at 48 hr after doses 1, 3 and 7, from two rats at 7 and 14 days after dose 7 and from five rats
at 21 days after dose 7. Total radioactivity in tissues was determined in these rats. One additional rat was used to determine the presence of
intact AR177 in tissues by HPLC and/or gel electrophoresis. Urine and
fecal samples were taken from five rats at each time point up to and
including day 21. The values that are presented are the means of these
replicate determinations. The blood, plasma, urine, feces and tissues
were stored frozen until analyzed.
Determination of 33P-AR177-associated
radioactivity in tissues, feces and body fluids.
Tissues, feces
and blood were homogenized in distilled water (20%, w/w), using a
Brinkman Polytron homogenizer, and solubilized with Soluene (Beckman).
Plasma, urine and aliquots of homogenized tissue, feces and blood were
placed in scintillation cocktail (Packard), and radioactivity was
determined in a scintillation counter (Beckman LS6000) using a quench
curve.
Calculation of the percentage of the total dose in each organ was based
on the actual weight of the excised organ. Calculations of the
percentage of the total dose in skeletal muscle, skin and bone marrow
were based on these tissues representing 50, 16 and 3% of the total
body weight, respectively (Burka et al., 1987).
Gel electrophoresis.
Tissues were homogenized for 10 sec
with 0.5 ml of Nonidet P-40 tissue extraction buffer/g of tissue in a
7-ml Kimble polyethylene vial containing one 4-mm glass bead in a
Wig-L-Bug (Crescent Dental Manufacturing Co., Lyons, IL). The Nonidet
P-40 tissue extraction buffer consisted of 0.2 M Tris, pH 7.5, 0.1 M
EDTA and 3% NP-40. The homogenate was incubated for 5 min at room
temperature and then rehomogenized in the Wig-L-Bug. After
homogenization, the tissues were incubated at 37°C for 24 hr to
extract 33P-AR177 from the tissues. The homogenate was then
transferred to a 2-ml microfuge tube and centrifuged for 10 min at
10,000 × g in an Eppendorf centrifuge. The
supernatant fraction was decanted and frozen in microfuge tubes for
later analysis.
The supernatant fraction (5 µl) from the tissue extraction or
untreated plasma or urine was mixed with sodium dodecyl sulfate sample
buffer (5 µl) and formamide (5 µl). The mixture was heated in a
boiling water bath for 2 to 3 min. Fifteen microliters were loaded onto
the 20% acrylamide/7 M urea gels, and the samples were electrophoresed
at 50 mA/gel until the bromphenol blue dye front was near the bottom of
the gel. All gels had nonradioactive AR177 run as a standard. This gel
electrophoresis system separates intact AR177 from n
1 and
n+1 species. To visualize nonradioactive AR177, the gels
were stained in Stains All (Sigma, St. Louis, MO) staining solution for
1 hr and then destained in 50% formamide for approximately 3 hr.
To prepare the gels for the detection of radioactivity, the gels were
incubated for 1 hr in 1% glycerol, 50% methanol and 10% glacial
acetic acid to help prevent cracking upon drying. The gels were dried
on Whatman no. 3 filter paper on a Bio-Rad gel drier.
The dried gels were exposed to the Fuji phosphor-imaging plate for 1 week and were then scanned on the Fujix BAS 1000 PhosphorImager. The
limit of sensitivity of the electrophoresis method was approximately 10 cpm/band, when the gels were exposed to the phosphor-imaging plate for
1 week. The images were stored on disk and annotated using the Fuji
MacBAS software.
Anion-exchange HPLC.
Tissues were homogenized for 10 sec
with 0.5 ml of Nonidet P-40 tissue extraction buffer/g of tissue in a
7-ml Kimble polyethylene vial containing one 4-mm glass bead in a
Wig-L-Bug (Crescent Dental Manufacturing Co.). The homogenate was
incubated for 5 min at room temperature and then rehomogenized in the
Wig-L-Bug. After homogenization, the tissues were incubated at 37°C
for 24 hr to extract 33P-AR177 from the tissues.
33P-AR177 is stable in serum and cells at 37°C for
several days (Bishop et al., 1996). The homogenate was then
transferred to a 2-ml microfuge tube and centrifuged for 10 min at
14,000 rpm in an Eppendorf centrifuge. The supernatant fraction was
decanted and frozen in microfuge tubes for later analysis by gel
electrophoresis or HPLC. The recovery of 33P-AR177 spiked
into control tissue homogenates was 60 to 70%.
The renal cortex, liver and spleen supernatants were chromatographed on
a strong anion-exchange HPLC column (Gen-Pak Fax column, 4.6 mm × 10 cm; Waters). The elution gradient consisted of buffer A (0.1 M Tris,
pH 12.0, 20% methanol) to buffer B (0.1 M Tris HCl, pH 12.0, 1.0 M
NaBr) over 60 min. The flow rate was 0.5 ml/min. 33P was
detected using an on-line radioactivity detector (Packard Radiomatic).
The chemiluminescence signal was subtracted as much as possible from
the overall signal, using the Radiomatic software, to obtain the true
33P signal. The presence of intact 33P-AR177 in
the HPLC-derived material was confirmed by collection of fractions in
an ISCO fraction collector, concentration of the fractions by speed
vacuuming and electrophoresis in a 20% acrylamide gel, as described
above.
Pharmacokinetic parameters.
Plasma, blood and tissue
pharmacokinetic values were calculated using RSTRIP II or PKAnalyst
(MicroMath, Salt Lake City, UT). Pharmacokinetic modeling of data from
both the single- and multiple-dose studies was performed on the mean
values of data from different sets of rats at each time point.
| |
Results |
Blood and plasma pharmacokinetics after a single i.v. bolus
injection of 33P-AR177.
After
a single i.v. dose of 33P-AR177, the disappearance of
drug from blood2 and plasma (fig.
1) fit a biexponential curve, which yielded an
AUC(0-
) of 24.2 µg-eq·hr/g
for blood and 13.5 µg-eq·hr/g for plasma (table 1).
The distribution half-lives for blood and plasma were similar (0.21 hr
for blood and 0.23 hr for plasma), and the elimination half-lives (
)
were long (367 hr for blood and 271 hr for plasma), based on total
radioactivity. The long elimination half-life of AR177 and slow
clearance (8.7 ml/hr from blood and 15.7 ml/hr from plasma) indicate
that there was slow elimination from rats. The 24.9-ml initial volume
of distribution in blood suggests that AR177 became rapidly distributed within the vascular compartment after dosing. The blood volumes of
distribution (4.6 or 4.1 liters, based upon postdistribution parameters
or steady-state conditions, respectively) showed that AR177 became
further distributed within the animals over 21 days.
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Fig. 1.
Pharmacokinetics of 33P-AR177 in rat
plasma after i.v. bolus injection. Data points are the means for
multiple rats at each time point, as described in "Materials and
Methods."
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TABLE 1
Blood and plasma pharmacokinetic parameters of AR177 equivalents after
single i.v. administration of 33P-AR177 (0.7 mg/kg) to male
Sprague-Dawley rats
Values are the mean data obtained from three to six rats per time
point, as described in "Materials and Methods."
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For approximately 1 hr after dosing, the plasma concentration of
33P-AR177 (microgram-equivalents per milliliter) was
approximately twice the concentration in blood, indicating limited
initial uptake of the drug into blood cells. However, at approximately
1 hr after dosing, the 33P-AR177 plasma/blood concentration
ratio began to decrease. Over time, the mean concentration of
33P-AR177 in plasma decreased more rapidly than that
observed in whole blood, possibly indicating uptake of AR177 into the
formed elements of the blood. This is also illustrated by an
approximately 1.4-fold difference between blood (367 hr) and plasma
(271 hr) terminal elimination half-lives.
Blood and plasma pharmacokinetics after multiple i.v. bolus
injections of 33P-AR177.
During multiple
dosing, concentrations of 33P-AR177 in
blood1 and plasma (fig. 2) were measured
before doses (Cmin) 2, 3, 4, 5, 6 and 7 and
after doses 1 and 7. With each subsequent dose, a steady increase in
Cmin was observed in both the blood and plasma, indicating that there was accumulation of the drug in the blood and
plasma. Maximum Cmin values of 0.32 µg-eq/g in
blood and 0.22 µg-eq/g in plasma were observed before the seventh of
seven doses. The observed whole-blood Cmin
values were consistent with theoretical values predicted from
single-dose data (fig. 1).
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Fig. 2.
Plasma concentration of 33P-AR177 after
multiple i.v. doses. Plasma was collected at various times after
multiple doses, as described in "Materials and Methods." Main plot,
blood was collected at six time points after dose 1, just before doses
2 to 6 and at eight time points after dose 7. Arrows, times of dosing.
Inset, plot of the plasma concentration of 33P-AR177 after
doses 1 and 7 over the first 8 hr (0.33 days). Values are the means for
multiple rats at each time point, as described in "Materials and
Methods."
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After the seventh dose, 33P-AR177 concentrations in blood
and plasma decreased in a biexponential decay pattern (fig. 2), similar to that observed after the single dose. Derived pharmacokinetic parameters were similar to those generated from the single-dose data.
The ratio of the single-dose
AUC(0-) to the seventh-dose
AUC(0-t), where t is
the dosing interval (0-48 hr), was in the range of one indicating
predictable accumulation in blood after multiple dosing.
AR177 concentrations in tissues after a single i.v. bolus injection
of 33P-AR177.
After single-dose
administration of 33P-AR177, radioactivity was
determined for up to 21 days in 14 tissues and was found to be widely
distributed (figs. 3 and 4). Peak
concentrations of 33P-AR177-associated radioactivity were
found in all tissues at the 8-hr time point after the single dose,
except for the testes (4 days) and thymus (32 hr) (table
2). Analysis of the AR177 tissue distribution showed
that the majority of the dose distributed to the liver (40%), bone
marrow (17%) and renal cortex (15%), at the peak at 8 hr after single
dosing. Analysis of the tissue concentration of 33P-AR177
showed that the highest values were achieved in the renal cortex (15.0 µg-eq/g), liver (7.4 µg-eq/g), bone marrow (3.9 µg-eq/g), mesenteric lymph node (3.0 µg-eq/g) and spleen (2.4 µg-eq/g) at 8 hr after single dosing. The half-lives in these tissues were as
follows: renal cortex, 231 hr (9.6 days); liver, 185 hr (7.7 days);
bone marrow, 883 hr (36.8 days); spleen, 739 hr (30.8 days); mesenteric
lymph node, 240 hr (10 days) (table 2).
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Fig. 3.
Concentrations of 33P-AR177 in the
renal cortex, liver, bone marrow, spleen mesenteric lymph node,
adrenals and renal medulla after a single i.v. dose of
33P-AR177. Starting on day 0, rats were dosed once with
33P-AR177 (0.7 mg/kg), as described in "Materials and
Methods." Arrow, time of the single dose. Tissues were taken at
various time points, as shown.  , renal cortex;  , liver;  , bone
marrow;  , spleen;  , mesenteric lymph node;  , adrenal; ×,
renal medulla. Values represent the mean of two to five rats per time
point.
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Fig. 4.
Concentrations of 33P-AR177 in the
thymus, lungs, testes, skeletal muscle, skin, brain and eyes after a
single i.v. dose of 33P-AR177. Starting on day 0, rats were
dosed once with 33P-AR177 (0.7 mg/kg), as described in
"Materials and Methods." Arrow, time of the single dose. Tissues
were taken at various time points, as shown. , thymus; , lungs;
, testes; , skeletal muscle; , skin;  , brain; ×, eyes.
Values represent the mean of two to five rats per time point.
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TABLE 2
Tissue pharmacokinetic parameters of AR177 equivalents after single
i.v. administration of 33P-AR177 (0.7 mg/kg) to male
Sprague-Dawley rats
Values are the mean data obtained from two to six rats per time point,
as described in "Materials and Methods." Data, taken from the
maximum plasma concentration (Cmax) to the last time point, were fit to a monoexponential equation. The correlation coefficients (r2) of the lines that were fit to the
data points were 0.97 (adrenal), 0.99 (bone marrow), 0.99 (liver), 0.94 (lymph node), 0.98 (renal cortex), 0.99 (skin), 0.99 (spleen), 0.98 (testes) and 0.97 (thymus). The Cmax values are
mean ± S.D. Pharmacokinetic parameters could not be determined
for the brain, lungs, renal medulla and skeletal muscle, because either
33P-AR177-associated radioactivity was still rising at 3 to 4 days after dosing (brain and skeletal muscle) or the data could not be
modeled.
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AR177 concentrations in tissues after multiple i.v. bolus
injections of 33P-AR177.
After multiple
dosing, concentrations of 33P-AR177-associated
radioactivity in tissues increased steadily (figs. 5 and
6). Peak concentrations of AR177 were found in all
tissues at 48 hr after the seventh (and last) dose, which was the first
sampling time after the last injection of 33P-AR177. After
the seventh dose, 33P-AR177-associated radioactivity in
tissues declined slowly over time. Gel electrophoresis of radioactivity
in the spleen, kidney and liver at 48 hr after the seventh dose of
33P-AR177 showed that approximately 90% of the
radioactivity represented intact drug (see results below). The total
percentage of the original dose decreased by approximately 48 ± 26% (mean ± S.D.) over 21 days in 12 of 14 tissues. These
tissues were the adrenals, bone marrow, eyes, liver, lungs, mesenteric
lymph node, renal cortex, renal medulla, skin, spleen, testes and
thymus. However, in 2 of 14 tissues, the brain and skeletal muscle, the
concentration of 33P-AR177-associated radioactivity
continued to rise, going from 0.02 and 0.08 µg-eq/g, respectively, at
8 hr after dose 7 to 0.08 and 0.11 µg-eq/g, respectively, at 21 days
after dose 7. It could not be determined whether the radioactivity
represented intact 33P-AR177 in the brain and skeletal
muscle, because of the low concentration of radioactivity. No
differences in the overall tissue distribution pattern were observed
after multiple dosing, compared with the single dosing. An average of
83% of the radioactive dose was recovered from the five rats over the
period of 34 days and seven doses.
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Fig. 5.
Concentrations of 33P-AR177 in the
renal cortex, liver, bone marrow, spleen mesenteric lymph node,
adrenals and renal medulla after multiple i.v. doses of
33P-AR177. Starting on day 0, rats were dosed once daily
every other day, for a total of seven doses, with 33P-AR177
(0.7 mg/kg), as described in "Materials and Methods." Arrows,
doses. Tissues were taken from rats at 48 hr after doses 1, 3 and 7 and
at 7, 14 and 21 days after dose 7. , renal cortex; , liver; ,
bone marrow; , spleen; , mesenteric lymph node; , adrenal; ×,
renal medulla. Values represent the mean of two to five rats per time
point.
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Fig. 6.
Concentrations of 33P-AR177 in the
thymus, lungs, testes, skeletal muscle, skin, brain and eyes after
multiple i.v. doses of 33P-AR177. Starting on day 0, rats
were dosed once daily every other day, for a total of seven doses, with
33P-AR177 (0.7 mg/kg), as described in "Materials and
Methods." Arrows, doses. Tissues were taken from rats at 48 hr after
doses 1, 3 and 7 and at 7, 14 and 21 days after dose 7. , thymus;
, lungs; , testes; , skeletal muscle; , skin; , brain;
×, eyes. Values represent the mean of two to five rats per time
point.
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Gel electrophoresis of plasma, urine and tissues.
To determine
whether radioactivity found in tissues of rats injected i.v. with
33P-AR177 represented intact AR177, gel electrophoresis was
used. Plasma, urine and selected tissues taken from
33P-AR177-dosed rats, after a single dose of
33P-AR177, were analyzed by gel electrophoresis, to
determine whether the radioactivity represented intact AR177. The
results showed that intact AR177 was present in the plasma, urine,
liver, kidney and spleen at all time points for which there was
sufficient radioactivity for gel analysis, based on co-migration with
nonradioactive AR177 standard. Although minor bands could be observed,
90% of the radioactivity in plasma, urine and tissues was found in a
band that co-migrated with nonradiolabeled AR177. Data for the liver
are shown in figure 7, taken at 2, 8, 24 and 48 hr after
a single dose of 33P-AR177, and are representative of
results in plasma, urine and other tissues.
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Fig. 7.
Analysis of 33P-AR177 in the liver by
gel electrophoresis, 48 hr after a single i.v. dose (0.7 mg/kg). Arrow,
position of the AR177 standard. The values represent the data from one
rat.
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The amount of radioactivity in the intact 33P-AR177 bands
was quantitated from the phosphor-images of the gels, as described in
"Materials and Methods." Figure 8 represents a
quantitative analysis and shows the counts per minute per band of
intact 33P-AR177 in the kidney, liver and spleen at 48 hr
after doses 1, 3 and 7 and 7 and 14 days after dose 7 of
33P-AR177. There is a close association between the results
of the extracted radioactivity in gels (fig. 8) and the results
obtained from counting the radioactivity in the same tissues (fig. 5).
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Fig. 8.
Quantitation of radioactivity in the renal cortex,
liver and spleen by gel electrophoresis, at various time points after
multiple doses 33P-AR177. The renal cortex, liver and
spleen were taken 48 hr after doses 1, 3 and 7 and 7 and 14 days after
dose 7 of 33P-AR177 (0.7 mg/kg). The tissues were extracted
and subjected to gel electrophoresis as described in "Materials and
Methods." The area of the radioactive spot was quantitated and the
counts per minute in the tissues were calculated at each time point. Arrows, doses. , renal cortex; , liver; , spleen. The values represent the data from one rat.
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HPLC of plasma, urine and tissues.
In addition to analysis of
radioactivity by gel electrophoresis, samples were analyzed by
anion-exchange HPLC. It was demonstrated that the radioactivity found
in the liver, taken as a representative example at 7 days after dosing,
represented intact 33P-AR177. Radioactivity extracted from
the liver (fig. 9A) eluted with the same retention time
as a 33P-AR177 standard (fig. 9B). Integration of the peak
from the liver sample indicated that it represented 95 to 100% intact
33P-AR177. Some chemiluminescence, contributed by
endogenous tissue substances, was seen in the void volume of the column
(before 5 min) and after the elution of the peak of radioactivity.
Thus, based on gel electrophoresis (90% intact) and HPLC (100%
intact) analysis, the radioactivity data used to calculate the
half-lives of 33P-AR177 in blood and plasma (table 1) and
tissues (table 2) conservatively represent approximately 90% intact
drug. This finding is consistent with the unusually long in
vitro stability of AR177 in serum that was reported by Bishop
et al. (1996).
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Fig. 9.
Analysis of 33P-AR177 in rat liver by
anion-exchange HPLC at 7 days after a single i.v. dose of
33P-AR177 (0.7 mg/kg). A, rat liver. B,
33P-AR177 standard.
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Excretion in urine and feces.
The major route of excretion was
urinary. In the single-dose experiment, the total amount of drug
excreted was 12.8% in the urine and 6.0% in feces (fig.
10) over 21 days after dosing. Gel electrophoresis of
the urine demonstrated that approximately 90% of the radioactivity
represented intact 33P-AR177 (data not shown). Gel
electrophoresis of the feces was not performed. In the multiple-dose
experiment, the total percentage of all seven doses excreted up to 21 days after dose 7 was approximately 30% in the urine and 10% in the
feces (data not shown).
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Fig. 10.
Urinary and fecal excretion after a single i.v.
dose of 33P-AR177. The urinary and fecal pharmacokinetic
data are derived from multiple rats at each time point after a dose of
0.7 mg/kg. , urinary percent cumulative excretion; , urinary
percent dose excreted per day; , fecal percent cumulative excretion;
 , fecal percent dose excreted per day. Values represent the
mean ± S.D.
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Discussion |
The pharmacokinetics of almost a dozen oligonucleotides have been
described in animals such as mice, rats and monkeys (Agrawal et
al., 1995), and the pharmacokinetics of two oligonucleotide have
been described in humans (Crooke, et al., 1994; Zhang
et al., 1995d). The half-lives of oligonucleotides vary
greatly, depending upon their sequence and modifications. The plasma
half-lives of total phosphorothioate oligonucleotides, which have
sulfur atoms substituted for at least one oxygen in the internucleotide linkages, have been reported to be the longest, with terminal plasma
half-lives of approximately 25 to 55 hr (Cossum et al., 1993, 1994; Zhang et al., 1995a,b,c; Srinivasan and Iversen,
1995). In contrast, the results of the present study show that AR177, a
17-mer oligonucleotide with only two phosphorothioate linkages, has
terminal half-lives of 367 hr in blood and 271 hr in plasma, far
greater than what has been previously reported for total
phosphorothioate or nonphosphorothioate oligonucleotides.
The reason for the low blood and plasma clearance (15.7 and 8.7 ml/hr)
and low excretion rate in urine and feces (total of 18.8% in urine
plus feces after 21 days after a single dose) is unknown but may be due
to the tight binding of AR177 to tissues. The evidence for this is
derived from observations that were made during the development of the
HPLC assay method for AR177. It was observed that AR177 elutes with a
salt gradient at approximately physiological pH in the absence of
tissues. After the injection of AR177 into rats, however, AR177 is
tightly associated with tissue components and cannot be separated
during anion-exchange HPLC at physiological pH. It was found to be
necessary to raise the pH of the HPLC elution buffer to 12 to
dissociate AR177 from tissues components, as reported in "Materials
and Methods." At pH 12, AR177 completely dissociates from tissues and
100% recovery is obtained. The identity of these components is
unknown. Although it is theoretically possible that the slow
elimination could be due to tight binding to plasma proteins, the large
steady-state volume of distribution argues against this possibility.
Thus, binding to tissues may be partially responsible for the long
half-life of AR177.
One of the positive features of AR177 that was found in this study was
its stability for long periods of time in blood, plasma and tissues.
The stability is probably due to the three-dimensional structure of the
molecule, in which the drug is folded onto itself by hydrogen-bonding
interactions between deoxyguanosine residues. Stabilization is
coordinated by a potassium ion in the center of the AR177 molecule
(Rando et al., 1995; Bishop et al., 1996). These
interactions result in a quartet structure, containing three loops and
four segments (Rando et al., 1995; Bishop et al.,
1996). As a result of this structure, which is the first to be reported for an oligonucleotide drug, AR177 is highly resistant to nucleases and
has a half-life of >4 days in serum in vitro and at least 48 hr intracellularly in vitro (Bishop et al.,
1996). Single-base mutations of AR177, with which the molecule could
not completely form the quartet structure, produced half-lives of only
3 to 7 min in serum. Although this quartet structure is responsible in large part for the resistance to nucleases, the phosphorothioate linkages at the 3- and 5-termini of AR177 also contribute to its
resistance to nucleases. This was demonstrated by showing that a total
phosphodiester version of AR177 has a shorter serum half-life than that
of AR177, although it has a serum half-life much longer than those of
nonquartet oligonucleotides (Bishop et al., 1996). Thus,
nuclease resistance may be partially responsible for the long half-life
of AR177.
Previous studies that have reported the tissue distribution of
oligonucleotides have shown that, in general, the highest
concentrations can be found in the liver and kidneys (Srinivasan and
Iversen, 1995; Zhang et al., 1995a,b,c). In agreement with
the results for other oligonucleotides, the concentration and
percentage of AR177 dose were highest in the liver and kidney.
Importantly for its potential utility as an anti-HIV drug, AR177 was
also found in high concentrations in lymphoid tissue, including both
the spleen and lymph node. Concentration of AR177 in these tissues is a
fortuitous characteristic of this drug, because lymphocytes are the
major repository of HIV and lymphocytes are concentrated in the spleen
and lymph nodes.
One question arising from these results is whether the long half-life
of AR177 will be reflected by a long duration of antiviral activity.
Although no results are yet available from antiviral studies in animals
or humans, studies in vitro support the idea that the long
half-life of AR177 causes long-lasting antiviral activity. In a study
reported by Ojwang et al. (1995), AR177 caused total
suppression of HIV-1 production in MT4 cells (a human lymphoid cell
line) in vitro for >21 days after washout of the drug and replacement with fresh culture medium. In contrast, resumption of HIV
viral synthesis occurred within 2 days after the removal of
azidothymidine in the same study.
Another question arising from these results is whether the long
residence time of AR177 in tissues will result in toxicity. Although no
results are yet available from phase I human studies, studies in
cynomolgus monkeys support the idea that the long half-life of AR177 is
not associated with toxicity. In a study reported by Wallace et
al. (1996b), AR177 did not cause any tissue damage or changes in
clinical chemistry when it was administered as a bolus i.v. injection
at doses up to 40 mg/kg every other day, for a total of 12 doses, to
cynomolgus monkeys. In a separate study, AR177 did not cause death,
cardiovascular toxicity or alterations in clinical chemistry in
cynomolgus monkeys receiving single doses up to 50 mg/kg as a 10-min
i.v. infusion (Wallace et al., 1996a), although there was a
transient and reversible prolongation of coagulation time at high
doses. Taken together, the data indicate that AR177 does not cause the
cardiovascular (Cornish et al., 1993; Galbraith et
al., 1994) or histopathological (Srinivasan and Iversen, 1995)
toxicities that are associated with total phosphorothioate oligonucleotides and it can be administered safely as an i.v. injection.
A third question regarding these findings is whether the blood, plasma
and tissue concentrations that were achieved in rats will be sufficient
to have an antiviral effect in humans. In vitro results have
previously shown that AR177 has an EC50 of approximately 0.2 µM (1.2 µg/ml) against many HIV-1 clinical isolates infected into human peripheral blood mononuclear cells (Ojwang et
al., 1995). Assuming that the minimum concentration of AR177 that
would be required to have antiviral activity in humans would be
approximately the in vitro EC50, and making the
assumption that AR177 is pharmacologically available in human blood,
plasma and tissues for antiviral activity, the results indicate that
therapeutic concentrations of AR177 can be achieved in many tissues
after single or multiple dosing. Furthermore, the therapeutic
concentration could possibly be sustained for several weeks after
single or multiple doses, based on the long tissue half-life reported
in this study. In lymphoid tissues, i.e., bone marrow,
spleen and mesenteric lymph node, the concentration of AR177 was
maintained at approximately 10, 7 and 3 times, respectively, the
in vitro EC50 of AR177 at 21 days after the
seventh and last dose of 0.7 mg/kg drug in rats. There are indications
that AR177 is probably taken up by cells in vivo. The higher
concentration of AR177 found in the blood vs. the plasma
indicates that the drug is taken up by blood cells. Studies with
radiolabeled AR177 have shown that the drug is readily taken up into
human cells in vitro (Bishop et al., 1996). Thus,
based on the pharmacokinetic results in rats, there is good reason to
expect that therapeutic concentrations of AR177 can be achieved, that
the therapeutic concentration can be maintained for several weeks in
humans and that the drug can be taken up into cells. Based on these
findings, it is believed that this drug has the potential to have
antiviral activity in humans. However, it is not known whether AR177 is available for antiviral activity in tissues. Clinical anti-HIV data are
not yet available.
In conclusion, AR177 exhibits long blood, plasma and tissue half-lives
after bolus i.v. administration and distributes widely to tissues in
rats. Of particular importance, because of the proposed anti-HIV
clinical indication, AR177 is distributed to lymphoid tissues in high
concentrations. The pharmacokinetic characteristics of AR177 in rats,
along with the low toxicity seen in cynomolgus monkeys, indicate that
AR177 has promising characteristics for the treatment of HIV infection
in humans. The every other day dosing protocol used in this study is
currently being used in a phase I study in humans.
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Abstract
Introduction
