Plasma Pharmacokinetics and Tissue Disposition of Novel
Dextran Methylprednisolone Conjugates With Peptide
Linkers in Rats
SUMAN PENUGONDA,1 HITESH K. AGARWAL,2 KEYKAVOUS PARANG,2 REZA MEHVAR1
1
Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center,
Amarillo, Texas
2
Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston,
Rhode Island
Received 2 June 2009; revised 4 August 2009; accepted 5 August 2009
Published online 24 September 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21934
ABSTRACT: The plasma and tissue disposition of two novel dextran prodrugs of
methylprednisolone (MP) containing one (DMP-1) or five (DMP-5) amino acids as linkers
were studied in rats. Single 5-mg/kg doses (MP equivalent) of each prodrug or MP were
administered intravenously, and blood and tissue samples were collected. Prodrug and
drug concentrations were quantitated using HPLC, and noncompartmental pharmaco-
kinetic parameters were estimated. Whereas conjugation of MP with dextran in both
prodrugs substantially decreased the clearance of the drug by 200-fold, the accumula-
tions of the drug in the liver, spleen, and kidneys were significantly increased by
conjugation. However, the extent of accumulation of DMP-1 in these tissues was
substantially greater than that for DMP-5. Substantial amounts of MP were regenerated
from both prodrugs in the liver and spleen, with the rate of release from DMP-5 being
twice as fast as that from DMP-1. However, the AUCs of MP regenerated from DMP-1 in
the liver and spleen were substantially higher than those after DMP-5. In contrast, in
the kidneys, the AUC of MP regenerated from DMP-5 was higher than that after DMP-1
administration. These data suggest that DMP-1 may be more suitable than DMP-5 for
targeting immunosuppression to the liver and spleen. ß 2009 Wiley-Liss, Inc. and the
American Pharmacists Association J Pharm Sci 99:1626 1637, 2010
Keywords: dextran prodrugs; macromolecular prodrugs; peptide linkers; methyl-
prednisolone; pharmacokinetics; tissue distribution; immunosuppression; liver
targeting
INTRODUCTION rejection1 4 that occurs in 60 80% of liver
transplant patients.2,5 However, this treatment
Intravenous mega doses (e.g., three 1-g pulses) of has been fatal in several cases6 11 and/or associat-
methylprednisolone (MP) succinate are currently ed with severe life-threatening toxicities related
the treatment of choice, and the most widely used to the cardiovascular system (cardiac arrest and
protocol, for the treatment of acute cellular arrhythmia),8,10 12 central nervous system
(seizure and blindness),6,13 16 severe infections
(viral and bacterial),2,3,5,17 or metabolic complica-
Correspondence to: Reza Mehvar (Telephone: [phone]
tions (hypokalamia).18 Therefore, a targeted
ext 337; Fax: [phone]; E-mail: [e-mail])
delivery of MP to the liver may afford adminis-
Journal of Pharmaceutical Sciences, Vol. 99, 1626 1637 (2010)
tration of smaller doses, resulting in prolonged
ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association
1626 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 3, MARCH 2010
PHARMACOKINETICS OF NOVEL DEXTRAN-METHYLPREDNISOLONE CONJUGATES 1627
local effects in the liver, without significant lengths (1 5 amino acids). In vitro studies24
toxicity to other organs and sudden death indicated that these prodrugs are subject to
observed with the current protocols.6 11 hydrolysis by lysosomal enzymes, with the rate
Recently, we developed a macromolecular of hydrolysis being proportional to the length of
prodrug of MP by attaching the drug to dextran the peptide linker. Therefore, it may be possible to
70 kDa through a succinate linker, producing two control the in vivo rate of release of MP from its
ester bonds at both ends of the linker. The dextran prodrugs by using different peptide
pharmacokinetics19 and systemic20 and local21 linkers. In the current study, we selected two
immunosuppressive activities of the conjugate second-generation dextran prodrugs of MP with
confirmed the feasibility of the use of dextrans for the shortest (one amino acid) and longest (five
delivery of MP to the liver and spleen. These amino acids) linkers for in vivo pharmacokinetic
studies demonstrated that the prodrug would studies in rats. Single doses of dextran MP
selectively accumulate and gradually release MP conjugates with one (DMP-1) or five (DMP-5)
in the liver and spleen, resulting in a more intense amino acid linkers (Scheme 1) or the parent drug
and sustained immunosuppressive activity in MP were administered to rats and their plasma
these organs, compared to administration of an pharmacokinetics and tissue disposition were
equal dose of the parent drug. Additionally, determined. Our hypothesis was that the in vivo
studies in a rat transplantation model showed rate of regeneration of MP from DMP-5 is faster
that this dextran prodrug of MP is more effective than that after the DMP-1 administration.
than the parent drug in preventing rejection of the
liver allograft.22 However, despite very high
accumulation of the prodrug in the liver and MATERIALS AND METHODS
spleen, the regeneration of MP (via enzymatic
hydrolysis of the ester bonds) in these tissues was Chemicals
very slow and incomplete.20,21 For example,
Dextran with an average Mw of 23,500 was
although the concentration of the conjugate in
obtained from Dextran Products, Ltd (Scarbor-
the spleen was still very high at 96 h after its
ough, Ontario, Canada). The degree of polydis-
administration (84.2 mg/g; 50% of the peak con-
persity of dextran was 2.3. 6a-Methylprednisolone
centration), no free MP was detected at this time,
(MP) was purchased from Steraloids (Newport,
resulting in the return of lymphocyte proliferation
RI). Internal standard (triamcinolone acetonide)
activity to the baseline levels. In fact, the ratio of
was purchased from Sigma (St. Louis, MO). For
the regenerated MP: 70 kDa succinic acid MP
chromatography, HPLC grade acetonitrile (EMD)
concentrations in the spleen progressively and
was obtained from VWR Scientific (Minneapolis,
rapidly declined with time after the administra-
MN). All other reagents were analytical grade and
tion of the conjugate.20 Similarly, despite the fact
obtained through commercial sources.
that the conjugate concentration in the liver at
DMP conjugates (Scheme 1) with methyl Gly
72 h after its administration was 40% of its peak
(mGly) (DMP-1) or mGly Gly Gly Gly Gly
concentration, it did not result in any MP
(DMP-5) were synthesized and characterized as
regeneration or immunosuppressive activities in
reported by us before.24 The degrees of MP
this organ at this time point.21 This was attributed
substitution (w/w) of the conjugates were 9.4%
to the type of linker (succinic acid) and steric
and 6.9% for DMP-1 and DMP-5, respectively,
hindrance exerted by the 70 kDa dextran.23 The
with purities of >90%.
slow and incomplete regeneration of MP from the
dextran 70 kDa succinic acid MP is undesirable
because the full potentials of the prodrug in terms
Animals, Dosing, and Sampling
of both intensity and duration of the effect are not
realized. Therefore, newer prodrugs of MP with All procedures involving animals used in this
faster and more controllable rate of release are study were consistent with the guidelines set by
desirable. the National Institute of Health (NIH Publication
In an effort to improve and control the release No. 85-23, revised 1985) and approved by our
profile of MP from its macromolecular prodrugs, Institutional Animal Care and Use Committee.
second-generation dextran MP prodrugs were Adult male Sprague Dawley rats were obtained
recently24 synthesized containing a lower Mw from Charles River Lab (Wilmington, MA)
dextran ( 25 kDa) and peptide linkers of varying and housed in a 12-h light dark cycle and
DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 3, MARCH 2010
1628 PENUGONDA ET AL.
Scheme 1. Chemical structures of methylprednisolone (MP), MP succinate
(MPS), dextran, and the two dextran prodrugs of MP with methyl (m) Gly (DMP-1)
or mGly Gly Gly Gly Gly (DMP-5) as linkers.
temperature-controlled facility prior to the study. dioxide, and blood (cardiac puncture) and tissues
A total of 64 animals were used for this study that were collected. The sampling times were 1 min
consisted of three groups. Two groups (21 rats/ and 0.5, 1.5, 3, 5, 12, and 24 h after DMP-1 or
group) were treated with DMP-1 or DMP-5 and DMP-5 injection, and 1, 10, 20, 40, 60, and 120 min
the third group (18 rats) was treated with MP. The after MP injection (n ź 3/time point). Also, total
remaining four rats were used as organ donors for urine output was collected from the 24-h groups
blank sample preparation. The mean standard after the DMP-1 or DMP-5 injections. Immedi-
deviation (SD) of the body weights of rats were ately after excision, the collected tissues were
250 12, 236 26, and 246 12 g for DMP-1, rinsed in ice-cold saline and blotted dry and
DMP-5, and MP groups, respectively. kept frozen until analysis. The blood was imme-
A single 5-mg/kg (MP equivalent) dose of either diately centrifuged in heparin-coated microcen-
MP or dextran prodrugs of MP were administered trifuge tubes to obtain plasma. One hundred
into the penile veins of rats under isoflurane microliters of plasma sample was transferred to a
anesthesia. The drugs were injected as bolus doses silanized microcentrifuge tube for DMP analysis.
over 15 s. At various times after dosing, different For MP analysis, 500 mL of plasma sample was
groups of animals were euthanized using carbon transferred to a silanized microcentrifuge tube
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 3, MARCH 2010 DOI 10.1002/jps
PHARMACOKINETICS OF NOVEL DEXTRAN-METHYLPREDNISOLONE CONJUGATES 1629
containing 100 mL of 10% acetic acid solution to under the plasma concentration time curve from
prevent DMP hydrolysis in vitro.25 All the time zero to the last measurable concentration
samples were stored at 808C until analysis. without (AUClast) and with (AUC0 1) extrapola-
tion to infinity, total body clearance (CL), initial
volume of distribution (V0), steady-state volume of
distribution (Vss), mean residence time (MRT),
Sample Analysis
maximum observed drug concentration (Cmax),
Before analysis, tissues were homogenized with
and time to reach Cmax (Tmax). The terminal
3 volumes of 2% (v/v) acetic acid, and the
elimination rate constant (lz) was calculated from
homogenate was used for the HPLC analysis of
the log-linear portion of plasma or tissue concen-
DMP and/or free MP. Previous studies have
tration time curves. The maximum concentra-
shown that the in vitro hydrolysis of DMP
tions of DMP or MP in plasma after the injection of
conjugates with peptide linkers in the liver
the conjugate or parent drug (C0) were assumed to
homogenates containing 2% acetic acid are
be the same as the concentrations at the first
negligible during sample preparation.25
sampling time (1 min). The concentrations of
The concentrations of free MP in plasma and
drugs in tissues were corrected28 for the contribu-
tissue homogenates were determined using a
tion from the residual blood using the volume
previously reported reversed-phase HPLC
fraction of blood (VB) in different organs; VB
method26 with a modified extraction procedure.
values of 0.0135, 0.061, 0.0459, 0.0572, 0.175, and
Briefly, to 240 mL of plasma and/or tissue homo-
0.321 were used for brain, heart, kidney, liver,
genates were added 600 mL of cold acetonitrile
lung, and spleen, respectively.29
containing 5 mg/mL of triamcinolone acetonide as
internal standard. After vortex mixing and
Statistical Analysis
centrifugation, the supernatant was evaporated,
and the analytes were first dissolved in 120 mLof
Because of the destructive nature of sampling, the
methanol, followed by the addition of 120 mL of
variance could not be estimated by normal
10 mM sodium acetate buffer (pH 4.5). After a
methods for most pharmacokinetic parameters
brief centrifugation, the resultant supernatants
except for Cmax, C0, V0, and the cumulative
were injected into the HPLC.
amount excreted unchanged in urine within
The concentrations of DMP in plasma were
24 h ðD24Þ, which are presented as mean SD.
U
measured using a slightly modified size-exclusion
Additionally, the variance of AUClast was esti-
HPLC (SEC) method described before.27 To
mated using the Bailer method, and the AUClast
precipitate proteins, 80 mL of cold methanol and
values are presented as mean SE.30
20 mL of 20% (v/v) perchloric acid were added to
Statistical comparisons were conducted either
100 mL of plasma. After a brief vortex mixing and
between DMP-1 and DMP-5 or among parent MP,
centrifugation, 150 mL of the supernatant was
MP regenerated from DMP-1, and MP regener-
transferred to a new microcentrifuge tube and
ated from DMP-5. For parameters with known
175 mL of a 0.2 M phosphate buffer (pH 7.0) were
variances (e.g., Cmax), statistical comparisons
added. Subsequently, a 100-mL aliquot was
among three groups were tested by one-way
subjected to the SEC method.
ANOVA, followed by Tukey s post hoc analysis,
The concentrations of DMP in tissue homo-
whereas unpaired, two-tailed t-test was used for
genates were quantitated according to a pre-
comparisons involving two groups (DMP-1 vs.
viously reported assay,19 with two minor
DMP-5). The statistical differences among dif-
modifications. Instead of ethanol, dextran con-
ferent groups in their AUC values were analyzed
jugates were precipitated with methanol, and the
using a two-sided Z-test after Bonferroni adjust-
acetonitrile proportion in the mobile phase was
ment for the appropriate number of comparisons,
35%, instead of 25%.
as described in detail before.19
Pharmacokinetic Analysis RESULTS
Noncompartmental pharmacokinetic parameters
Plasma Pharmacokinetics
were calculated using WinNonlin 5.2.1 (Pharsight
Company, Mount View, CA). The pharmacoki- The plasma concentration time courses of
netic parameters obtained included the area DMP-1, DMP-5, and MP after intravenous doses
DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 3, MARCH 2010
1630 PENUGONDA ET AL.
Table 1. The Pharmacokinetic Parameters of the
Parent MP, DMP-1, and DMP-5 after Single 5 mg/kg
(MP Equivalent) Intravenous Doses of the Parent Drug
or the Prodrugs
Injected Drug
MP DMP-1 DMP-5
Measured Analyte MP DMP-1 DMP-5
C0 (mg/mL) 2.13 0.27 83.4 1.7 92.5 8.6
V0 (mL/kg) 2370 286 60.0 1.2 54.4 5.2
Vss (mL/kg) 2470 365 89.5
AUClast (mg h/mL) 0.468 0.028 77.8 4.5 74.7 2.5
AUC0 1 (mg h/mL) 0.480 100 80.3
CL (mL/min/kg) 174 0.832 1.04
MRT (h) 0.237 7.32 1.44
T1/2 (h) 0.183 9.02 1.57
 a 15.4 6.1 24.7 8.5
D24 (% dose)
U
a
Not determined.
Figure 1. The plasma concentration time courses of
DMP-1, DMP-5, and MP after intravenous administra- mGly Gly Gly Gly Gly (DMP-5) linker resulted
tion of single 5 mg/kg (MP equivalent) doses of the in a substantial (170- to 210-fold) reduction in the
prodrugs or the parent drug to rats. The symbols and
plasma clearance of the drug. However, the CL
bars represent the mean and SD values, respectively
values of the two conjugates were relatively close
(n ź 3 for each point).
(Tab. 1). Additionally, dextran conjugation caused
substantial reductions in the overall extent of
distribution of the drug, as reflected in both V0 and
of 5 mg/kg (MP equivalent) of the prodrugs or the
VSS. However, the magnitudes of the decrease in
parent drug are presented in Figure 1. The plasma
the volume of distribution of MP as a result
concentrations of MP after the administration
of conjugation were much smaller than those
of the parent drug were relatively low and not
detectable beyond 60 min after the drug admin- observed for the CL (Tab. 1). Furthermore,
although the initial volumes of distribution (V0)
istration. In contrast, both DMP-1 and DMP-5
of the two conjugates were similar, the VSS of
demonstrated very high concentrations, which
DMP-1 was 4-fold larger than that for DMP-5.
were quantifiable for at least 5 h. Whereas the
The higher VSS of DMP-1 was also reflected in the
plasma concentrations of DMP-1 were also
longer terminal half-life and MRT for this
measurable at 12 h in all the rats, no detectable
conjugate (Tab. 1). Twenty-four hours after the
traces of DMP-5 were found at this time point
drug injection, 15.4% and 24.7% of the dose of
(Fig. 1).
After the administration of DMP-1, no regen- DMP-1 and DMP-5, respectively, were recovered
unchanged in the urine (Tab. 1).
erated MP was detectable in all the plasma
samples, except for the 1-min sample, which
contained an MP concentration of 0.180
0.020 mg/mL. The plasma concentrations of the
Tissue Disposition
regenerated MP after DMP-5 injection, which
ranged from 2.94 0.83 mg/mL at 1 min to The hepatic concentration time profiles of the
0.239 0.122 mg/mL at 3 h (data not shown), were administered MP, DMP-1, and DMP-5 and MP
higher than those after the administration of the regenerated in vivo from DMP-1 and DMP-5 are
parent drug (Fig. 1), suggesting in vitro regenera- presented in Figure 2. Additionally, the respective
tion of MP from DMP-5 during sample collection, pharmacokinetic parameters are presented in
storage, and/or analysis. Table 2. Following the administration of the
The pharmacokinetic parameters of MP, DMP- parent drug MP to rats, the maximum liver
1, and DMP-5 are presented in Table 1. Conjuga- concentrations of MP (1.14 0.45 mg/g) occurred
tion of MP to dextran using the mGly (DMP-1) or within the first sample (1 min), and, thereafter,
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 3, MARCH 2010 DOI 10.1002/jps
PHARMACOKINETICS OF NOVEL DEXTRAN-METHYLPREDNISOLONE CONJUGATES 1631
DMP-5 were 760- and 220-fold, respectively,
larger than that of the parent drug (Tab. 2). Also,
although the Cmax values of the regenerated MP
after the administration of both prodrugs were
similar to that after the administration of the
parent drug ( p > 0.05), they occurred at a later
time, and the decline in the hepatic concentrations
of the regenerated MP after DMP-1 and DMP-5
were much more sustained than that after the
parent drug (Tab. 2 and Fig. 2). Therefore, the
hepatic AUC0 1 of regenerated MP after DMP-1
and DMP-5 were 14- and 10-fold, respectively,
higher than that after the administration of the
parent drug (Tab. 2). We also estimated the liver/
plasma AUC ratios after the parent drug or
prodrug administration as a measure of liver
targeting. Whereas the ratio for DMP-5 (2.98) was
Figure 2. The hepatic concentration time courses of
close to that for the parent drug (2.29), the ratio
the administered DMP-1, DMP-5, and MP and the MP
for DMP-1 (8.40) was 4-fold higher than that for
regenerated from DMP-1 and DMP-5 after intravenous
the parent drug (Tab. 2).
administration of single 5 mg/kg (MP equivalent) doses
The splenic concentration time profiles and
of the prodrugs or the parent drug to rats. The symbols
disposition parameters of all the administered
and bars represent the mean and SD values, respec-
drugs (MP, DM-1, and DM-5) and MP regenerated
tively (n ź 3 for each point).
from the two prodrugs are presented in Figure 3
and Table 3, respectively. Compared with the
the concentrations declined very rapidly (Fig. 2) parent drug MP, the concentrations of the two
with a terminal half-life of 0.708 h (Tab. 2). In prodrugs in the spleen were very high, with the
contrast, the hepatic concentrations of both Cmax values occurring much later. Similar to the
prodrugs DMP-1 and DMP-5, after the adminis- liver profiles (Fig. 2), DMP-1 achieved much
tration of equivalent MP doses, were much higher higher concentrations in the spleen, when com-
than those of the parent drug and declined pared with DMP-5 (Fig. 3). Both DMP-1 and DMP-
relatively slowly with terminal half lives of 5 released MP in the spleen (Fig. 3). Although the
15.0 and 9.07 h, respectively (Fig. 2 and Tab. 2). Cmax values of the regenerated MP were similar
Indeed, the hepatic AUC0 1 values of DMP-1 and after DMP-1 and DMP-5 administration, the Tmax
Table 2. The Hepatic Disposition Parameters of the Parent MP, DMP-1, and DMP-5 and the MP Regenerated from
DMP-1 or DMP-5 after Single 5 mg/kg (MP Equivalent) Intravenous Doses of the Parent Drug or the Prodrugs
Injected Drug
MP DMP-1 DMP-5
Measured Analyte MP MP DMP-1 MP DMP-5
Cmax (mg/g) 1.14 0.45 1.02 0.41 35.6 4.49a 1.09 0.69 18.1 1.44a
Tmax (h) 0 1.5 1.5 3 1.5
AUClast (mg h/g) 0.927 0.088b 7.63 1.10b 560 42a 9.57 0.87b 143 6a
AUC0 1 (mg h/g) 1.10 15.0 842 10.8 239
Liver/plasma (AUC ratio) 2.29  c 8.40  c 2.98
MRT (h) 1.02 17.2 21.6 9.79 13.0
T1/2 (h) 0.708 12.3 15.0 7.47 9.07
a
Significant difference between DMP-1 and DMP-5.
b
Significant differences between parent MP and MP regenerated from DMP-1 or between parent MP and MP regenerated from
DMP-5.
c
Not estimated because of the lack of accurate estimate of plasma AUC.
DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 3, MARCH 2010
1632 PENUGONDA ET AL.
quantitation for most samples. Additionally, no
detectable peaks of DMP-1 or DMP-5 were found
in any of the brain samples. However, relatively
significant concentrations of DMP-1 and DMP-5
were found in the kidneys (Fig. 4 and Tab. 4).
Similar to the pattern in the liver and spleen, the
DMP-1 AUC in the kidney was several-fold larger
than that of DMP-5. However, in contrast to
the liver and spleen, the higher and sustained
concentrations of DMP-1 in the kidneys were
associated with relatively low levels of regener-
ated MP, which were only detectable within the
first 30 min of DMP-1 injection (Fig. 4).
DISCUSSION
Figure 3. The splenic concentration time courses of
the administered DMP-1, DMP-5, and MP and the MP
Our recent in vitro studies24 indicated that
regenerated from DMP-1 and DMP-5 after intravenous
dextran prodrugs of MP with linkers containing
administration of single 5 mg/kg (MP equivalent) doses
1 5 (methyl) Gly residues were degraded by rat
of the prodrugs or the parent drug to rats. The symbols
and bars represent the mean and SD values, respec- liver lysosomal fractions at a rate directly
tively (n ź 3 for each point). proportional to the length of the linker. The
present work was then undertaken to study the
in vivo pharmacokinetics, tissue distribution, and
was much longer and the AUC was substantially extent of MP regeneration of the two prodrugs of
higher after the DMP-1 injection (Tab. 3). MP with the shortest (DMP-1) and longest (DMP-
Furthermore, the spleen/plasma AUC ratios, a 5) linkers. Although both prodrugs significantly
measure of targetability to the spleen, increased altered the pharmacokinetics and tissue disposi-
from 2.90 for the parent drug to 54.8 and 10.9 for tion of MP qualitatively in a similar manner,
DMP-1 and DMP-5, respectively (Tab. 3). substantial differences were observed between
In terms of tissues other than the liver and the two prodrugs, most notably in their extent of
spleen, the concentrations of DMP-1 and DMP-5 tissue distribution and regeneration of MP
in the heart and lungs were below the level of (Figs. 2 4 and Tabs. 2 4).
Table 3. The Splenic Disposition Parameters of the Parent MP, DMP-1, and DMP-5 and the MP Regenerated from
DMP-1 or DMP-5 after Single 5 mg/kg (MP Equivalent) Intravenous Doses of the Parent Drug or the Prodrugs
Injected Drug
MP DMP-1 DMP-5
Measured Analyte MP MP DMP-1 MP DMP-5
Cmax (mg/g) 9.94 0.81a 3.43 0.07a 145 30b 3.62 1.32a 39.4 5.5b
Tmax (h) 0 12 5 3 3
AUClast (mg h/g) 1.30 0.06a 54.3 1.0a,c 2250 145b 21.5 2.1a,c 423 15.4b
AUC0 1 (mg h/g) 1.39 89.2 5500 25.7 873
Spleen/plasma (AUC ratio) 2.90  d 54.8  d 10.9
MRT (h) 0.162 24.7 41.7 11.3 35.7
T1/2 (h) 0.202 13.3 26.5 10.6 25.3
a
Significant differences between parent MP and MP regenerated from DMP-1 or between parent MP and MP regenerated from
DMP-5.
b
Significant difference between DMP-1 and DMP-5.
c
Significant difference between MP generated from DMP-1 and MP generated from DMP-5.
d
Not estimated because of the lack of accurate estimate of plasma AUC.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 3, MARCH 2010 DOI 10.1002/jps
PHARMACOKINETICS OF NOVEL DEXTRAN-METHYLPREDNISOLONE CONJUGATES 1633
extent of tissue distribution suggest that the
linker affects the physicochemical properties of
the prodrug that are critical in its distribution.
Alternatively, the observed differences between
DMP-1 and DMP-5 in their VSS may have
been influenced by their different degrees of
MP substitution (9.4% vs. 6.9%, respectively).
Whereas dextrans are extremely water soluble,
MP is very lipophilic. Therefore, a 27% lower MP
content of DMP-5, relative to DMP-1, may have
contributed, in part, to a lower lipophilicity of
DMP-5 and a smaller VSS for this conjugate.
Additionally, because of the higher MP content,
the dose of dextran backbone was 27% lower after
DMP-1 injection. However, it is unlikely that a
27% difference in the dose of the carrier would be
responsible for the 4- to 10-fold observed higher
Figure 4. The kidney concentration time courses of
accumulation of DMP-1 in the tissues (Tabs. 2 4).
the administered DMP-1, DMP-5, and MP and the MP
Although less accumulated in the tissue than
regenerated from DMP-1 and DMP-5 after intravenous
DMP-1, DMP-5 regenerated MP faster than did
administration of single 5 mg/kg (MP equivalent) doses
DMP-1 in all the tissues. This is demonstrated by
of the prodrugs or the parent drug to rats. The symbols
comparisons of the AUCs of the regenerated MP
and bars represent the mean and SD values, respec-
with those of their prodrugs in each tissue. In the
tively (n ź 3 for each point).
liver, the AUC of the regenerated MP comprised
1.7% of the AUC of the prodrug after DMP-1
In all the studied tissues, DMP-1 achieved administration and 4.5% of the AUC of the
significantly higher concentrations (Figs. 2 4) prodrug after DMP-5 administration (Tab. 2).
and AUCs (Tabs. 2 4), compared with those Similarly, in the spleen the ratios were 1.62% and
observed after DMP-5 administration, suggesting 2.94% after the DMP-1 and DMP-5 injections,
a larger extent of distribution for this prodrug. respectively (Tab. 3). In the kidneys, the ratio was
This difference was also reflected in a fourfold the highest for DMP-5 (9.25%) among all the
larger VSS for DMP-1, estimated from the studied tissues, whereas virtually no release of
plasma concentration data (Tab. 1). The signifi- MP was observed after DMP-1 administration
cant differences between the two prodrugs in their (Tab. 4). These data suggest that the in vivo
Table 4. The Kidney Disposition Parameters of the Parent MP, DMP-1, and DMP-5 and the MP Regenerated from
DMP-1 or DMP-5 after Single 5 mg/kg (MP Equivalent) Intravenous Doses of the Parent Drug or the Prodrugs
Injected Drug
MP DMP-1 DMP-5
Measured Analyte MP MP DMP-1 MP DMP-5
Cmax (mg/g) 0.779 0.274a 0.823 0.179b 24.0 0.3 4.26 2.13a,b 36.2 14.9
Tmax (h) 0 0 0 0.5 0.5
AUClast (mg h/g) 0.195 0.018a  c 337 6d 6.10 0.98a 47.1 6.5d
AUC0 1 (mg h/g) 0.285  c 1020 7.17 77.5
Kidney/plasma (AUC ratio) 0.591  c 10.3  e 0.953
MRT (h) 0.614  c 59.6 2.23 6.60
T1/2 (h) 0.5  c 41.5 2.47 6.98
a
Significant difference between parent MP and MP regenerated from DMP-5.
b
Significant difference between MP regenerated from DMP-1 and MP regenerated from DMP-5.
c
ND: Not determined because only two kidney samples showed measurable concentrations of MP.
d
Significant difference between DMP-1 and DMP-5.
e
Not estimated because of the lack of accurate estimate of plasma AUC.
DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 3, MARCH 2010
1634 PENUGONDA ET AL.
release of MP from the prodrugs is faster for DMP- liver, spleen, and kidneys, respectively, compared
5 than DMP-1, an observation that is in complete with DMP-5. It should be noted the true index of
agreement with the in vitro release data reported tissue targetability for prodrugs is the tissue/
by us previously.24 plasma AUC ratios of the regenerated drug. This
The faster in vivo release of MP from its dextran parameter was not estimated in our study because
prodrug with the longer peptidyl linker (DMP-5) of the lack of regeneration of MP in the plasma
is in agreement with the literature data on a after DMP-1. However, it is clear that based on
dextran prodrug of a camptothecin analog this parameter, the selectivity of DMP-1 for
(T-2513), containing peptidyl linkers.31 Harada targeting to the liver and spleen would have been
et al.31 studied the in vivo release of T-2513 in the even more dramatic (because of the very low
liver and tumor of rats bearing Walker-256 plasma concentrations of MP regenerated from
carcinomas at 6 h after a single 1-mg/kg dose of DMP-1, compared with DMP-5). However, the
T-2513 conjugated to carboxymethyldextran (Mw same may not be true for the kidneys because in
of 130 kDa) via linkers comprising of one to five this tissue, the concentrations of MP regenerated
Gly amino acids. Similar to our studies, an from DMP-5 were higher than those after DMP-1
increase in the length of the linker progressively (Fig. 4). Overall, our data indicate that although
increased the in vivo rate of release of T-2513. releasing MP at a slower rate, DMP-1 is superior
However, they did not detect any release of T-2513 to DMP-5 in terms of targeting MP to the liver and
from the prodrug with Gly linker. This is in spleen.
contrast to a relatively significant release of MP In addition to the MP prodrug developed by us,
from DMP-1 in the liver in our studies (Fig. 2). at least three other examples are available in the
This difference could be attributed to the differ- literature with regard to the use of peptide
ences in the two prodrugs in the chemistry of the linkers for the attachment of drugs to dextran
bond between the drug and the carrier and/or carriers.31 37 In all of these examples, carbox-
the much larger size of the dextran carrier used ymethyldextran, a negatively charged derivative
in their study (130 kDa), compared with ours of dextran, with high Mw was used as a carrier
( 25 kDa). with a peptide linker with three,31 33 four,34,35
It may be argued that the lower concentrations or six36,37 amino acids to deliver anticancer
of DMP-5 in the tissues, compared with DMP-1 drugs camptothecin derivatives31 35 or metho-
(Figs. 2 4), are due to a faster release of MP from trexate36,37 to the tumor. These prodrugs exhibit
this prodrug and not because of differences in the increased circulation time, relative to that of the
tissue accumulation between the two prodrugs. parent drug, allowing higher accumulation of the
Although a faster MP release might have con- anticancer drugs in the tumors, where they
tributed to the lower tissue concentrations of gradually release the parent drug, resulting in
DMP-1, in particular in the kidneys, it cannot higher efficacy and/or lower toxicity in animal
explain the substantial differences between the and/or human studies. Our prodrugs intended for
tissue AUCs of DMP-1 and DMP-5 in the liver and the delivery of MP to the liver and spleen differ
spleen. This is because in both the liver (Tab. 2) from these anticancer prodrugs mainly in that we
and spleen (Tab. 3), the AUC of the generated MP used a neutral dextran, instead of the negatively
after DMP-1 was higher than that after the charged carboxymethyldextran used for the deliv-
administration of DMP-5. Therefore, the differ- ery of the anticancer drugs. This was because
ences in the tissue accumulation of the two previous studies38 have shown that negatively
prodrugs, at least in the case of liver and spleen, charged dextrans have a lower accumulation in
are likely due to a selective accumulation of DMP- the liver, compared with neutral dextrans. There-
1 in these tissues. fore, neutral dextrans are more suitable than
The tissue/plasma AUC ratio is a more appro- negatively charged dextrans for delivery to the
priate index of tissue targeting, compared with liver, which is intended for our MP prodrug.
the absolute AUC values in the tissues. Based The two dextran prodrugs of MP tested in the
on these AUC ratios, both dextran prodrugs of present in vivo study (DMP-1 and DMP-5) are
MP are superior to the parent drug in terms of part of a second-generation dextran prodrugs of
targeting MP to the liver (Tab. 2), spleen (Tab. 3), MP that were recently synthesized and charac-
and kidneys (Tab. 4). Further, a comparison of the terized in vitro.24 A major difference between
ratios for the two prodrugs indicate that DMP-1 these prodrugs and a prodrug of MP developed
has 3-, 5-, and 11-fold higher selectivity for the earlier (dextran 70 kDa succinic acid MP) is the
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 3, MARCH 2010 DOI 10.1002/jps
PHARMACOKINETICS OF NOVEL DEXTRAN-METHYLPREDNISOLONE CONJUGATES 1635
use of amino acid/peptide linker (instead of a (378C) with half-lives of 24.5 and 4.67 h, respec-
succinate linker) in the new prodrugs. The peptide tively. Additional studies25 also showed that the
linkers were selected to circumvent the inflexible addition of acetic acid to the plasma samples
and slow rate of MP regeneration observed with during the sample preparation and storage
dextran 70 kDa succinic acid MP.19,20 Indeed, prevents in vitro degradation of the prodrugs in
whereas the ratio of the AUC of the regenerated plasma. Although blood samples in our study were
MP over that of the prodrug in the liver was only centrifuged immediately to separate plasma
0.60% for dextran 70 kDa succinic acid MP,19 the before the addition of acetic acid and storage,
corresponding ratios were 1.8% and 4.5% for the MP concentrations in the plasma samples of
DMP-1 and DMP-5, respectively (Tab. 2). Simi- DMP-5-treated animals were substantially higher
larly, the ratios for DMP-1 and DMP-5 in the than the corresponding concentrations after the
spleen (1.6% and 2.9%, respectively; Tab. 3) were administration of the same dose of the parent drug
higher than that for dextran 70 kDa succinic (see Results Section). Pharmacokinetically, the
acid MP (0.93%).19 These data indicate that not AUC of a drug after the administration of its
only is the rate of MP regeneration faster with the prodrug cannot be higher than that after the
new prodrugs, but also can be controlled by administration of the same dose of the parent
varying the length of the linker. drug, suggesting that despite our precautions
The second major difference between the newly DMP-5 was converted to MP in the blood in vitro
developed prodrugs described here and dextran during centrifugation. This is a plausible expla-
70 kDa succinic acid MP is the lower Mw of the nation because in vitro degradation of even 1 2%
new prodrugs (25 kDa instead of 70 kDa). Our of the very high concentrations of the prodrug in
previous work with dextran carriers demon- the plasma (Fig. 1) can easily result in the values
strated that high Mw dextrans (Mw 20 kDa) observed after DMP-5. Consequently, we did not
preferentially accumulated in the liver and report here the plasma AUC values of MP after
spleen, whereas low Mw dextrans (e.g., Mw of DMP-5 administration. This problem did not
4 kDa) were rapidly excreted into the urine occur with DMP-1 because of much higher
without achieving significant concentrations in stability of this prodrug, compared with DMP-5,
the liver or spleen.28 Among dextrans with a Mw in in blood.24 Nevertheless, our data clearly show
the range of 4 150 kDa, dextran with a Mw of that DMP-1 does not regenerate MP to a
70 kDa achieved the highest accumulation in the significant degree in the blood (in vivo or
liver. Therefore, in our initial studies designing a in vitro), whereas DMP-5 is prone to the release
prodrug of MP, we used the 70 kDa dextran as a of MP in the blood both in vivo and in vitro.
carrier. However, the elimination of dextran An ideal macromolecular prodrug of a drug with
70 kDa is strictly via nonrenal pathways,28 its site of action in the tissue is expected to remain
making it susceptible to nonlinearity in its intact in the circulation, enter the tissues and cells
pharmacokinetics.23,39 Therefore, in designing of interest, and release the drug intracellularly. If
the newer dextran prodrugs of MP, we selected the drug is released prematurely in the circula-
a dextran with a Mw of 25 kDa, which is expected tion, the prodrug will lose its targeting effect,
to be subject to some degree of linear renal resulting in no change in drug exposure. Our
excretion,39 in addition to a significant accumula- tissue data for the liver (Fig. 2 and Tab. 2) and
tion in the liver.28 Therefore, another major spleen (Fig. 3 and Tab. 3) for both DMP-1 and
difference in the pharmacokinetics of the newer DMP-5 conjugates clearly show that the tissue
prodrugs (DMP-1 and DMP-5) and dextran exposure to released MP was much higher,
70 kDa succinic acid MP is that in contrast to compared with the parent drug administration.
no renal excretion for dextran 70 kDa succinic This suggests that the MP present in these tissues
acid MP, between 15% and 25% of the adminis- after the conjugate administration is most likely
tered dose of DMP-1 or DMP-5 was recovered in regenerated from the conjugate intracellularly.
the 24-h urine of rats as intact prodrug (Tab. 1). This is consistent with previous studies40,41
These data suggest that the potential for the long- demonstrating that dextrans enter the cells by
term, undesired accumulation of the carrier endocytosis, ending up in lysosomes, which
dextran in the tissues is less for these novel contain enzymes capable of degrading peptide
dextran prodrugs of MP. bonds such as those in DMP-1 and DMP-5.24
Our recent in vitro studies24 showed that DMP- In conclusion, our data show that the novel
1 and DMP-5 are degraded in the rat blood in vitro dextran prodrugs of MP with amino acid/peptide
DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 3, MARCH 2010
1636 PENUGONDA ET AL.
linkers are suitable for delivery of the drug to the 6. Stubbs S, Morrell R. 1973. Intravenous methylpred-
nisolone sodium succinate: Adverse reactions
liver and spleen for the purpose of immunosup-
reported in association with immunosuppressive
pression. In agreement with the reported in vitro
therapy. Transplant Proc 5:1145 1146.
data,24 our current in vivo study shows that the
7. McDougal BA, Whittier FC, Cross DE. 1976. Sud-
regeneration of MP from the prodrug with the five
den death after bolus steroid therapy for acute
amino acid linker (DMP-5) is much faster than
rejection. Transplant Proc 8:493 496.
that from the prodrug with the one amino acid
8. Bocanegra TS, Castaneda MO, Espinoza LR, Vasey
linker (DMP-1) in all the tissues. However, the
FB, Germain BF. 1981. Sudden death after methyl-
AUC of MP regenerated from DMP-1 is substan-
prednisolone pulse therapy. Ann Intern Med 95:
tially higher than that regenerated from DMP-5
122.
in the liver and spleen. In contrast to the liver and
9. Gardiner PV, Griffiths ID. 1990. Sudden death
spleen, minimal regeneration of MP occurred in after treatment with pulsed methylprednisolone.
Br Med J 300:125.
the kidneys after DMP-1 administration. There-
10. Moses RE, McCormick A, Nickey W. 1981. Fatal
fore, DMP-1 may be more suitable than DMP-5 for
arrhythmia after pulse methylprednisolone ther-
targeting immunosuppression to the liver and
apy. Ann Intern Med 95:781 782.
spleen. Further pharmacodynamic studies are
11. Guillen EL, Ruiz AM, Bugallo JB. 1998. Hypoten-
required to determine the effects of these prodrugs
sion, bradycardia, and asystole after high-dose
on the local immunosuppression in the liver after
intravenous methylprednisolone in a monitored
liver transplantation.
patient. Am J Kidney Dis 32:E4.
12. McLuckie AE, Savage RW. 1993. Atrial fibrillation
following pulse methylprednisolone therapy in an
adult. Chest 104:622 623.
ACKNOWLEDGMENTS
13. Ayoub WT, Torretti D, Harrington TM. 1983. Cen-
tral nervous system manifestations after pulse
This study was supported by a grant from the
therapy for systemic lupus erythematosus. Arthri-
National Institute of General Medical Sciences
tis Rheum 26:809 810.
of NIH (R01 GM069869). The authors acknowl-
14. Suchman AL, Condemi JJ, Leddy JP. 1983. Seizure
edge the use of WinNonlin program as part of a
after pulse therapy with methyl prednisolone.
Pharsight Academic Licensing Program. Arthritis Rheum 26:117.
15. Cerilli J, Miller JA. 1972. The effect of massive
pulse steroid therapy on the water content of the
rat brain. Transplantation 14:403 405.
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DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 3, MARCH 2010