Plasma-mediated release of morphine from synthesized prodrugs

Article abstract of DOI:10.1016/j.bmcl.2010.08.098

Two morphine prodrugs ('PDA' and 'PDB') were synthesized and the kinetics of esterase-mediated morphine release from these prodrugs were determined when incubated with plasma from different animal species. Morphine was rapidly released from PDA by all spe

Full text of DOI:10.1016/j.bmcl.2010.08.098

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6250–6253
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Plasma-mediated release of morphine from synthesized prodrugs
⇑
Thommey P. Thomas , Baohua Huang, Ankur Desai, Hong Zong, Xue-min Cheng, Alina Kotlyar,
⇑
Pascale R. Leroueil, Thomas Dunham, Abraham van der Spek, Brent B. Ward, James R. Baker Jr.
Michigan Nanotechnology Institute for Medicine and Biological Sciences, Department of Internal Medicine, Division of Allergy, University of Michigan, 9220 MSRB III, Box 0648,
Ann Arbor, MI 48109, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Two morphine prodrugs (‘PDA’ and ‘PDB’) were synthesized and the kinetics of esterase-mediated mor-
phine release from these prodrugs were determined when incubated with plasma from different animal
species. Morphine was rapidly released from PDA by all species plasma with the maximum reached
within 5–10 min; the released morphine was biologically active as determined by an in vitro cAMP assay.
The morphine was released from PDB at a slower and species-dependent rate (mouse > rat > guinea
pig > human). Morphine’s release from PDB appeared to be mediated by carboxyl esterases as the release
was inhibited by the carboxyl esterase inhibitor benzil. PDA nor PDB induce cytotoxicity in the neuronal
cell lines SK-NSH and SH-SY5Y. The carboxyl and amino functional moieties present on the linker por-
tions of PDA and PDB, respectively, may facilitate their conjugation to nanoparticles to tailor morphine
pharmacokinetics and specific targeting. These studies suggest the potential clinical utility of these pro-
drugs for morphine release at desired rates by administration of their mixture at selected ratios.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 24 June 2010
Revised 18 August 2010
Accepted 19 August 2010
Available online 24 August 2010
Keywords:
Morphine
Prodrugs
Plasma
Esterase
Cytotoxicity
Morphine (MP) is an established standard in the treatment of
acute and chronic pain.1 However, morphine has a relatively short
plasma half-life (1–3 h) associated with a correspondingly limited
duration of analgesic effect.2 Under certain circumstances, longer-
acting morphine preparations may be desirable. Longer-acting,
sustained-release oral morphine preparations are available, how-
ever, certain medical conditions, such as inability to swallow, vom-
iting, bowel obstruction, etc., may prevent the oral administration
of morphine.
Longer-acting and sustained-release morphine preparations for
parenteral administration (e.g., intravenously, intramuscularly,
and subcutaneously) do not exist. Frequent parenteral injections
or infusions may not be available, feasible, be uncomfortable or
inconvenient. Therefore, morphine prodrugs with long half-life
which can release morphine in a slow, sustained fashion may be use-
ful in the treatment of acute and chronic pain (e.g., from acute inju-
ries sustained in remote, desolate places without healthcare
facilities, and cancer), avoiding the need for frequent parenteral
morphine injections. Moreover, combinations of morphine prodrugs
tailored for fast-, intermediate-, and long-acting release may be
ideal for alleviating a range of acute and chronic pain conditions.
Because the free 3-OH group of morphine is a prerequisite for
its analgesic activity,3,4 different 3-substituted prodrugs have been
synthesized and shown to have varying plasma release kinetics
and biological activity.5–7 Our overall goal is to synthesize 3-substi-
tuted morphine prodrugs with linkers containing open functional
groups which allow conjugation to nanoparticles, such as dendri-
mers,8 to change its pharmacokinetics and enable specific target-
ing. We report an abbreviated view of the synthesis of two
morphine prodrugs and their morphine release kinetics in the
presence of plasma from different animal species.
The structures of the synthesized morphine prodrugs,
designated as prodrug A (PDA) and prodrug B (PDB), are shown
in Figure 1. Their synthesis is summarized in the Supplementary
data section and in Fig. S1. The proton NMR spectra analysis
revealed that the substitutions have taken place at 3-OH of the
morphine (Fig. S2).
Ultra performance liquid chromatography (UPLC)9 (Waters Inc.)
was utilized for measurements of morphine, PDA and PDB. UPLC
has the capability to perform rapid (<10 min) and reproducible
chromatographic separations using small sample volumes com-
pared to conventional high performance liquid chromatography
(HPLC) techniques,10 for example, ꢀ1–3
L for UPLC versus ꢀ30–
l
50 lL for HPLC. The term ‘morphine’ refers to the free morphine
alkaloid base (Malinkrodt, etc.) unless otherwise stated.
Baseline UPLC profiles were obtained for phosphate buffered
saline (PBS), morphine and PDA in esterase de-activated plasma.
Plasma was precipitated by the addition of two volumes of 10%
DMSO in acetonitrile (ANDM). This pretreated plasma was then
spiked with morphine or PDA, and the supernatant was subjected
to UPLC. ANDM allows both deproteinization as well as the release
of protein-bound MP.11
⇑
Corresponding authors. Tel.: +1 734 615 1569; fax: +1 734 615 2506 (T.P.T.).
E-mail addresses: thommey@umich.edu (T.P. Thomas), jbakerjr@umich.edu
(J.R. Baker).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.098

T. P. Thomas et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6250–6253
6251
300
250
200
150
100
50
Mouse
Rat
Guinea Pig
Human
0
0
60
120
180
240
300
360
420
Time, Minutes
Figure 3. The kinetics of the release of morphine from PDA by plasma from four
species. PDA (250 M) was incubated with 50% plasma in PBS at 37 °C, then aliquots
Figure 1. Chemical structures of morphine prodrugs A and B.
l
were withdrawn at different time intervals, mixed with 2 volumes of ice-cold
ANDM solution and centrifuged to remove the protein precipitate. The supernatant
was subjected to UPLC analysis and concentrations of the released morphine were
calculated from standard curve generated for morphine.
Baseline UPLC values of ANDM treated human plasma showed
major peaks at 0.65, 0.89, 2.2, 2.4, 5.1, 5.4, 8.2, and 10.7 min.
(Fig. 2). PDA in ANDM treated human plasma eluted out at
4.3–4.4 min, while morphine appeared as dual peaks, with a major
peak at 1.65–1.75 min, and a shoulder or a minor peak at 1.60–
1.65 min. UPLC peaks of ANDM treated human plasma did not
overlap the elution time peaks of morphine or PDA. When mor-
phine and PDA were diluted in PBS and then subjected to similar
UPLC analyses (data not shown), elution times for morphine and
PDA were similar to those observed in ANDM treated human plas-
ma, indicating that ANDM treated plasma components did not
influence the elution profile of these two drugs.
conditions for up to 24 h (data not shown), the rapid release of
morphine was likely due to plasma components such as plasma
esterases. A comparison of the effects of rat and guinea pig plasma
and serum on PDA showed that they both induced the release of
morphine with equal potency (data not shown), showing that nei-
ther the blood coagulation proteins nor the added anticoagulant in
the plasma samples influence the hydrolytic process.
The released morphine was tested for biological activity by
determining its known
l-opioid receptor mediated signal trans-
To quantify the amount of morphine released, a standard curve
was generated by injection of different amounts of morphine in
PBS into the UPLC column and quantifying the areas under the
peak curves (1.6–1.75 min.) for each concentration. The morphine
peak area was linear from 10 nM to 3.5 mM morphine injected
(Fig. S3), with an R value of 0.99.
The effect of plasma from different animal species on the kinet-
ics of the release of morphine from PDA was determined. Most
morphine was released within 5–10 min in the presence of plasma
(Fig. 3). Concurrent with the increase in the morphine peak, over
>90% of the PDA peak (eluting at 4.3–4.4 min) disappeared at all
incubation conditions. While PDA was stable in PBS under similar
duction property to decrease the cellular cAMP content.12 We uti-
lized the human neuronal cell line SK-NSH known to have cell
surface
l
-opioid receptors expressed.13 cAMP was quantified by
a chemiluminescent assay using a cAMP standard curve (Fig. S4).
Morphine released from PDA significantly inhibited forskolin-stim-
ulated cAMP (forskolin is an activator of adenylate cyclase which
increases the basal cellular cAMP) (Fig. 4). PDA incubated in PBS
under identical conditions failed to significantly inhibit cAMP
production. These results show that the morphine released from
the prodrug is biologically active.
70
60
50
40
30
20
10
0
Control
PDA + PBS
0.039
0.017
0.011
0.005
-0.001
0.017
0.011
0.005
-0.001
0.029
0.019
0.009
-0.001
PDA + HP
4.1
4.3
4.5
1.5
1.6
1.7
1.8
*
MP
PDA
-
-
+
+
-
+
FSK:
0.0
2.0
4.0
6.0
8.0
10.0
12.0
Figure 4. The effect of vehicle- and plasma-incubated PDA on forskolin-stimulated
cAMP in SK-NSH cells. PDA (0.8 mM) was incubated with PBS or 50% human plasma
(HP) for 24 h. SK-NSH cells plated in 24-well plates were changed to serum-free
medium and incubated with the control- or plasma-incubated PDA solutions
(equivalent to 5 lM prodrug) for 15 min, followed by 15 min incubation with 5 lM
forskolin (FSK) or its vehicle. The cells were rinsed, lysed, and the cAMP content was
Time, Minutes
Figure 2. UPLC profile of human plasma spiked with morphine (MP) or PDA.
Human plasma was first mixed with ice-cold ANDM, then spiked with morphine
(green), PDA (red), or PBS (black) followed by centrifugation. The protein-free
supernatant from each of these separate sample mixtures was subjected to UPLC,
using conditions described in the Supplementary data.
determined from a cAMP calibration curve (Fig. S2), using the ‘GraphPad Prism’
*
software. p <0.05 versus control and ‘PDA + PBS’.

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T. P. Thomas et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6250–6253
245
A
A
0.019
0.015
0.011
0.007
0.003
-0.001
spiked MP
195
Mouse Plasma
60'
Rat Plasma
145
95
45
-5
30'
15'
Guinea Pig Plasma
C, 0' & 5'
Human Plasma
Human BCE
Human Albumin
1.60
1.64
1.68
1.72
1.76
1.80
0
60
120
180
240
300
360
420
Time, Minutes
Time, Minutes
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
B
0'
295
245
195
145
95
B
15'
Human Albumin
Human BCE
Human Plasma
30'
Guinea Pig Plasma
Rat Plasma
60'
180'
45
Mouse Plasma
4.45
4.50
4.55
Time, Minutes
4.60
-5
0
60
120
180
240
300
360
420
Time, Minutes
Figure 5. UPLC profile of the release of morphine from PDB by rat plasma, showing
the time-dependent increase in morphine (A) and the corresponding decrease in
PDB (B). The control plasma profile is indicated as ‘C’. The ‘spiked MP’ shown (solid
Figure 6. The kinetics release of morphine from PDB by plasma from different
animal species, purified human serum BCE (sigma B4186, 0.5 U/ml), and human
serum albumin (sigma 8763, 25 mg/ml). (A) Morphine release quantified from the
peak area of morphine. (B) The corresponding disappearance of PDB quantified from
the peak area of PDB. Other experimental details are as given for Figure 2 and in the
Supplementary data.
black) is rat plasma spiked with morphine (150 lM) following treatment with
ANDM, under similar conditions. The unlabeled graphs (coinciding with the 600) are
those obtained for 180 and 360 min. Other details are as given for Figure 2.
Subsequently, the kinetics of morphine’s release from prodrug
B (PDB) by plasma from different species was tested. Figure 5 illus-
trates the UPLC profile obtained for the time-dependent release of
morphine by rat plasma. The PDB eluted out at 4.4–4.6 min, while
the released morphine did so at 1.6–1.8 min. With rat plasma,
about 50% of the morphine release occurred in 30 min, reaching
a maximum within 60 min. We also observed the concurrent,
time-dependent increase of a second peak at 4.22–4.32 min, which
probably represented the aromatic linker cleaved off from PDB
(see Fig. 1 and Fig. S5).
The rate of release of morphine from PDB by plasma of different
species is shown in Figure 6. Among the different plasmas tested,
the rate of hydrolysis was highest with mouse plasma, followed
by rat > guinea pig > and human plasma. The higher hydrolytic
activity in the rodents may be due to the presence of large amounts
of serum carboxylesterases, as compared to insignificant levels
observed in the human serum.14 In contrast to our observation, a
previous study has shown that human plasma hydrolyzed mor-
phine-3-propionate at a significantly higher rate than rat plasma,15
suggesting different ester substrates may have different specificity
and esterase hydrolytic activity.
ꢀ4% in 3–6 h. Under similar assay conditions, 50% human plasma
released ꢀ4% morphine in 15 min and ꢀ20% in 6 h. These results
show that PDB is a poor substrate for BCE and HSA, however, other
carboxyl esterases16 or aryl esterase17 may participate in PDB’s
hydrolysis. These findings are similar to those measured for hydro-
lysis of the prodrug irinotecan by human plasma,18 but different
120
100
80
60
40
60'
30'
20
10'
0
0
20
40
60
80
100
120
Butyryl choline esterase (BCE) and human serum albumin (HSA)
have been suggested to play a role in ester hydrolysis.14 We also
determined the effects of 25 mg/ml HSA and 0.5 U/ml purified hu-
man serum BCE, concentrations equivalent to 50% of their known
levels in human serum.14 HSA released ꢀ1% of morphine from
PDB in 15 min, which reached a maximum of ꢀ2% in 3–6 h. The
BCE released ꢀ2.5% morphine from PDB in 15 min, reaching to
[Benzil], µM
Figure 7. Dose-dependent inhibition of plasma-mediated release of MP from PDB,
by the carboxyl esterase inhibitor benzil. PDB was incubated with rat plasma in the
presence of different concentrations of benzil for 100, 300, or 600, and the released
morphine was quantified by UPLC analysis. Other conditions are as given for
Figure 6. The data is given as the percent of maximal morphine release obtained for
the control sample at 600.

T. P. Thomas et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6250–6253
6253
relevant concentrations. XTT assay21 of PDA and PDB showed that
these prodrugs are not cytotoxic up to 1 M in the neuronal cell
Table 1
Comparison of the rate of hydrolysis of PDB by different plasmas, human BCE and HSA
l
lines SK-NSH and SH-SY5Y (Fig. 8). This suggests that neither the
prodrugs nor the linker moiety that is released in the serum-con-
taining medium during the 3 days incubation is cytotoxic.
In summary, we have demonstrated differential plasma hydro-
lytic activities for two novel morphine prodrugs carrying chemi-
cally functional 3-substituted groups. Future studies will focus on
the pharmacokinetics of the conjugates administered individually
or as a mixture. The prodrugs can be further utilized for conjuga-
tion to nanoparticles such as the neutralized and non-cytotoxic
dendrimers,22,23 which may allow improved drug solubility,
payload, pharmacokinetics, and controlled release.
Plasma/enzyme MP release rate MP release
Adult blood Projected
source
(nmol/min/ml) rate
g/min/ml)
(vol, ml)
release
(l
(mg/h/adult)
Mouse
Rat
Guinea Pig
Human
BCE
20.9
4.2
2.9
1.1
0.8
0.3
5.97
1.19
0.83
0.31
0.23
0.10
1.5
25
0.54
1.8
25
1.2
4000
74.5
HSA
The data is derived from the 0–15 min time points at which time the rates were
linear for all samples (Fig. 6). The reaction mixture contained an initial amount of
37.5 nmol of PDB and 150 ll plasma/enzyme samples in a total volume of 300 ll.
The rates given are per ml of plasma, or enzyme samples at known levels in human
plasma.
Acknowledgment
This project has been funded with Federal funds from the
Defense Advanced Research Projects Agency –DOD, under award
W911NF-07-1-0437.
120
100
Supplementary data
80
SK-NSH, PDA
SK-NSH, PDB
SH-SY5Y, PDA
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.08.098.
60
SH-SY5Y, PDB
References and notes
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