Anhydride prodrugs for nonsteroidal anti-inflammatory drugs

Article abstract of DOI:10.1023/A:1022214919481

Purpose. The objective of this study was to synthesize anhydride prodrugs for prolong action to shield the carboxylic acid group from irritative effects and to temporary hydrophobize the drug so that it becomes accessible to aqueous media when the anhydride residue is hydrolyzed. Methods. Ibuprofen, a nonsteroidal anti-inflammatory agent, was used as a representative drug for anhydride derivatization. Mixed anhydrides of ibuprofen with fatty acids of different chain length were prepared by reacting acid chloride derivatives with the corresponding acid in the presence of acid acceptor and two-phase reaction. Mixed anhydrides were also prepared by dehydration reaction using acetic anhydride and anhydride interchange of symmetric anhydrides. The analgesic effects of mixed anhydride prodrugs were tested using nonsteroidal anti-inflammatory drug rat paw edema model. In vitro degradation of mixed anhydrides and drug release were monitored by high-performance liquid chromatography. Results. Ibuprofen was bound to aliphatic and aromatic acids via an anhydride bond in high reaction yields (>85%) with high mixed anhydride content (>80%). The mix anhydride was purified by chromatography and stored at 4°C to minimize conversion into the symmetric anhydride. These anhydride derivatives hydrolyzed at different time intervals depending on the hydrophobicity of conjugated acid. In vivo testing of the ibuprofen anhydride derivatives for analgesic effect indicated an extended action of the drug for over 24 h as a function of the fatty acid chain length. Conclusion. This study demonstrates the promise of anhydride prodrugs for extending drug action and shielding the carboxylic acid group.

Full text of DOI:10.1023/A:1022214919481

Pharmaceutical Research, Vol. 20, No. 2, February 2003 (© 2003)
Research Paper
The general approach to overcome these limitations is
esterification of the carboxylic acid groups to produce lipo-
philic and nonirritating prodrug forms, provided that the par-
ent bioactive agent can be released from the prodrug at its
sites of activity (1). However, several aliphatic and aromatic
esters of carboxylic acid drugs are not sufficiently labile in
vivo to ensure a sufficiently high rate and extent of prodrug
conversion. For example, ethyl esters of naproxen (2) and
fenbufen (3) have lower anti-inflammatory activity relative to
the free acids because of the resistance of esters to hydrolyze
in vivo. In the field of angiotensin-converting enzyme inhibi-
tors, ethyl esters of enalaprilic acid and its derivatives im-
proved their oral bioavailability. Plasma enzymes do not hy-
drolyze these esters fast enough and the necessary conversion
of ester to free acid predominantly takes place in the liver (4).
As a result of this reason, liver function may be a very im-
portant determinant for the bioactivation of enalapril and its
therapeutic effect. In another consideration, ester derivative
prodrugs have been found cleavable enzymatically to release
its bioactive forms and, thus, are dependent on the enzymatic
activity, which may vary among individuals, or even in the
same individuals at various times during the day or in various
sites where the drug is administered. This fact may result in a
large variation of drug bioavailability (5–7).
To overcome the above-mentioned limitations, anhy-
dride derivatives of carboxylic acid-bearing drugs are sug-
gested. Unlike the ester bond used in prodrugs, the anhydride
bond is more susceptible to hydrolysis and decomposition of
its carboxylic acid counterparts in a predictable rate and pat-
tern and is less sensitive to enzymolysis than the esters or
amides (8). Studies on polyanhydride as drug carriers show
that the anhydrides degrade in a controlled fashion and are
biocompatible with the human body tissues, including the
brain (9,10). Interestingly, not much attention has been given
to the formation of prodrugs based on the hydrolytically de-
gradable anhydride bonds, although biodegradable polymers
based on these bonds have been developed and used as erod-
ible carriers for drugs both in animals and in humans (8–10).
This article presents a new class of prodrugs based on
mixed anhydrides of carboxylic acid bearing drugs character-
ized by possessing a high susceptibility to undergo hydrolytic
degradation and at the same time providing ample possibili-
ties for varying the water and lipid solubilities of the deriva-
tives. The structure of the anhydride prodrug is illustrated
using ibuprofen as drug and linear aliphatic acids as carriers.
Anhydride Prodrugs for Nonsteroidal
Anti-Inflammatory Drugs
Osnat Shaaya,1 Amir Magora,2 Tzviel Sheskin,1,3
Neeraj Kumar,1 and Abraham J. Domb1,4
Received August 13, 2002; accepted November 1, 2002
Purpose. The objective of this study was to synthesize anhydride
prodrugs for prolong action to shield the carboxylic acid group from
irritative effects and to temporary hydrophobize the drug so that it
becomes accessible to aqueous media when the anhydride residue is
hydrolyzed.
Methods. Ibuprofen, a nonsteroidal anti-inflammatory agent, was
used as a representative drug for anhydride derivatization. Mixed
anhydrides of ibuprofen with fatty acids of different chain length
were prepared by reacting acid chloride derivatives with the corre-
sponding acid in the presence of acid acceptor and two-phase reac-
tion. Mixed anhydrides were also prepared by dehydration reaction
using acetic anhydride and anhydride interchange of symmetric an-
hydrides. The analgesic effects of mixed anhydride prodrugs were
tested using nonsteroidal anti-inflammatory drug rat paw edema
model. In vitro degradation of mixed anhydrides and drug release
were monitored by high-performance liquid chromatography.
Results. Ibuprofen was bound to aliphatic and aromatic acids via an
anhydride bond in high reaction yields (>85%) with high mixed an-
hydride content (>80%). The mix anhydride was purified by chro-
matography and stored at 4°C to minimize conversion into the sym-
metric anhydride. These anhydride derivatives hydrolyzed at differ-
ent time intervals depending on the hydrophobicity of conjugated
acid. In vivo testing of the ibuprofen anhydride derivatives for anal-
gesic effect indicated an extended action of the drug for over 24 h as
a function of the fatty acid chain length.
Conclusion. This study demonstrates the promise of anhydride pro-
drugs for extending drug action and shielding the carboxylic acid
group.
KEY WORDS: mixed-anhydride; prodrugs; ibuprofen; NSAID; pro-
long-action.
INTRODUCTION
A wide variety of compounds having carboxylic acid
groups are biologically active, for example, the nonsteroidal
anti-inflammatory drugs (NSAIDs), such as naproxen, ibu-
profen, indomethacin, and diclofenac; antibiotics, such as am-
picillin and cefmetazole; and herbicides, such as Tordon, En-
dothal, and Amiben. The presence of free acid groups in these
compounds produces local irritation on interaction with mu-
cosal tissues, and at the same time they ionize at physiological
pH, which makes the drug poorly absorbed through biologic
membrane.
Prodrugs will degrade slowly by hydrolysis in a predictable
and controlled fashion, eliciting the pharmacological response
characteristic of the acids from which they are derived. These
prodrugs are characterized to be less irritating to topical and
gastric or intestinal mucosal membranes. Prodrugs of carbox-
ylic acid agents may provide increased biomembrane trans-
port so that the parent drugs are more bioavailable from the
site of administration, such as the gastrointestinal tract, the
rectum, the blood–brain barrier, the skin, or the eye. In ad-
dition, anhydride prodrugs may temporarily modify the physi-
1 Department of Medicinal Chemistry and Natural Products, School
of Pharmacy, Faculty of Medicine, The Hebrew University of Jeru-
salem, Jerusalem, Israel 91120.
2 Division of Identification and Forensic Science, Israel Police Na-
tional Headquarters, Jerusalem, Israel 91906.
3 Current addresses: Taro Research Institute Ltd., Haifa Bay, Israel
26110.
4 To whom correspondence should be addressed. (e-mail: adomb@
md.huji.ac.il)
205
0724-8741/03/0200-0205/0 © 2003 Plenum Publishing Corporation

206
Shaaya et al.
cal properties of a drug to allow new formulations. For ex- using an oil pump (170°C, 0.5 mm Hg) the crude yellow liquid
ample, some ibuprofen derivatives as shown in this report are was extracted with hexane (90% yield). IR (film cast) 1800
liquid at room temperature, which allows their formulation in cm−1 (Cl-CסO) 1H NMR: 7.22 (q, 4H); 4.12 (q, 1H); 2.53 (D,
soft gelatin capsules. We report here on the synthesis of 2H); 1.88 (Octa, 1H); 1.62 (D, 3H); and 0.95 ppm (D, 6H).
mixed anhydrides of ibuprofen with various fatty acids, their
analysis, in vitro degradation, and preliminary in vivo antiin-
flammatory properties.
Preparation of Ibuprofen Sodium
Ibuprofen (10 g) was dissolved in double-distilled water
with the addition of sodium hydroxide solution (1 N). The
MATERIALS AND METHODS
Ibuprofen was a gift from Ethyl Corp. (NJ, USA). Acetic
anhydride, propionic anhydride, hexanoic anhydride, octa-
noic anhydride, lauric anhydride, benzoic anhydride, and
their respective acids and acid chlorides were purchased from
Aldrich (Milwaukee, WI, USA). Thionyl chloride and acetic
anhydride were distilled freshly under nitrogen before use,
and all solvents were of analytical grade. High-pressure liquid
chromatography (HPLC)-grade acetonitrile and dichloro-
methane were purchased from LabScane (Dublin, Ireland).
HPLC apparatus used for acids and anhydrides was com-
posed of an HP 1050 (Hewlett-Packard, Palo-Alto, CA,
USA) modular system, including a diode array UV-detector
and an IBM compatible data-system HPCHEM attached with
a ThinkJet printer. Injections were made using a Rheodyne
(Cotati, CA, USA) injection valve with a 20-␮L loop.
Samples (10 ␮L in acetonitrile) were eluted through a C8
column (Supelcosil 8, Supelco, PA, USA) using mixtures of
acetonitrile and water at a flow rate of 1 mL/min. Preparative
HPLC for the separation of mixed anhydrides was conducted
on a Giloson system composed of 305, 306, and 806 Mano-
metric Model pump system, a Rheodyne (Cotati, CA) injec-
tion valve with a 5-mL loop, and a 117 UV detector. MODcoI
columns, 25 × 2.54 cm with packing media CER-1481 or 1489
(MODcoI, St. Louis, MO, USA) was used with acetonitrile as
eluent.
solution was lyophilized under reduced pressure (10 mm Hg)
at –50°C for 34 h to obtain a white powder (85% yield). IR
(film cast) 1710 cm−1 (CסO) 1H NMR: 7.22 (q, 4H); 4.03 (q,
1H); 2.53 (D, 2H); 1.88 (Octa, 1H); 1.62 (D, 3H); and 0.95
ppm (D, 6H).
Synthesis of Mixed Anhydrides
Mixed anhydrides of ibuprofen and fatty acids (C3–C18)
were synthesized by the following methods:
Schotten Baumann Reaction
In this method, an acid and the corresponding acid chlo-
ride react in the presence of a tertiary amine as acid acceptor.
Pyridine, triethylamine, and crosslinked poly(4-vinylpyridine)
(PVP; Reillex® 402, Reilly Industries, NJ, USA) were used as
acid acceptors. The acid, acid chloride, and the amine (1:1.1:3
molar ratio) were mixed in dichloromethane (10% by weight,
10 mL) at 0°C with constant stirring of one hour, and evap-
orated to dryness. The oily residue was weighed and analyzed
1
by HPLC, IR, and H NMR.
In a typical reaction, ibuprofen (4.1 mmol) was added
with constant stirring to a solution of hexanoyl chloride (3.7
mmol) and PVP (10 mmol pyridine equivalents) in dry di-
chloromethane (10 mL) immersed in an ice bath. After one
hour of stirring at 0°C, the reaction mixture was quickly fil-
tered and washed with 5-mL portions of 0.1 N HCl followed
by distilled water and dried over magnesium sulfate. The
clear solution thus obtained was evaporated to dryness using
a nitrogen stream and analyzed. IR (cm−1) 1800, 1735; 1H-
NMR 7.21 (d, 2H), 7.05 (d, 2H), 3.70 (m, 1H), 2.45 (dd, 3H),
2.35 (t, 2H), 1.85 (m, 2H), 1.63 (m, 2H), 1.50 (m, 1H), 1.33 (dd,
4H), 0.88 (d, 9H). Ibuprofen mixed anhydrides with longer
fatty acids show similar IR and H NMR spectra with the peak
integration at 1.33 ppm increases to reflect the increase in the
number of methylenes in the fatty chain.
Infrared (IR) spectroscopy (Anelect Instruments FT-IR
model fx-6160) was performed for the drugs and anhydride
samples cast on NaCl plates using CH2Cl2 as solvent. Kontron
Instruments (Uvikon model 930) was used to record the UV
spectra. 1H NMR spectra (CDCl3/TMS/d/ppm) were ob-
tained by using 300-MHz spectrometer (Varian, CA, USA).
Ibuprofen Analysis
Quantitative analysis of ibuprofen was performed in a
C18 Lichospher 100 column (Merck, Dorstadt, Germany, 250
× 4 mm, 5 ␮m) using a 70:30 v/v mixture of acetonitrile:water
as mobile phase at a flow rate of 1 mL/min. Ibuprofen was
detected by UV at 270 nm, and its typical retention time was
4.4 min.
Two-Phase Reaction
Preparation of Ibuprofen Anhydride
Ibuprofen (10 g) was refluxed in acetic anhydride (100
mL) for 30 min followed by the evaporation of acetic anhy-
dride to dryness and the residue was distilled under vacuum
(0.3 mm Hg at 170°C) to yield (70%) a viscous liquid of
symmetric ibuprofen anhydride. IR (cm−1) 1812, 1748; 1H-
NMR 7.05 (d, 8H), 3.65 (m, 2H), 2.45 (dd, 4H), 1.85 (m, 2H),
1.42 (dd, 6H), 0.92 (d, 12H).
A two-phase reaction was performed between benzoyl
chloride (organic phase) and ibuprofen sodium (aqueous me-
dium) with and without the presence of pyridine N-oxide
(PNO) as phase transfer agent. Ibuprofen sodium (0.1 M) in
deionized water (10 mL) was mixed with a dichloromethane
solution (10 mL) containing hexanoyl chloride (0.1 M), PNO
(6 × 10−6 M), and naphthalene as internal standard for HPLC
analysis. Both phases were stirred vigorously at room tem-
perature and samples were withdrawn at the time interval of
Preparation of Ibuprofen Acid Chloride
Ibuprofen was dissolved in thionyl chloride (2 g in 5 mL) 5 min, dried over magnesium sulfate, evaporated to dryness,
and refluxed for 2 h. After distilling off the thionyl chloride and analyzed by HPLC.

Anhydride Prodrugs for NSAIDs
207
Anhydride Interchange
Symmetric ibuprofen-anhydride obtained above was dis-
solve in dichloromethane along-with a symmetric fatty acid
anhydride like hexanoic anhydride and stirred for 30 min at
room temperature followed by evaporation to dryness. The
oily residue thus obtained was dissolved in minimal amount of
acetonitrile and separated by preparative HPLC to isolate the
ibuprofen mixed anhydride.
Dehydration Method
Equivalent amounts of ibuprofen and hexanoic acid were
added to a boiling solution of acetic anhydride and refluxed
for 1 h followed by evaporation to dryness. The resulting
residue was dissolved in acetonitrile (0.5 mL) and immedi-
ately separated by preparative HPLC to isolate the mixed
anhydride.
In Vitro Degradation of Ibuprofen–Fatty
Acid-Mixed Anhydrides
Hydrolytic degradation of ibuprofen-fatty acid mixed an-
hydrides was studied by suspending a sample of the viscous
solution in 10 mL of 0.1 M phosphate buffer pH 7.4 at 37°C
with constant shaking (100 rpm). After a fixed time interval,
an aliquot of the suspension (5 mL) was withdrawn and ibu-
profen concentration was determined by HPLC using citrate
buffer (0.01M, pH 3): acetonitrile (60:40 v/v) mixture as mo-
bile phase with UV detection at 264 nm.
In Vivo Analgesic Activity: Randall-Selitto Assay in Rats
Analgesic activity of ibuprofen mixed anhydrides was
evaluated using the Randall Selitto NSAID rat paw edema
model. For this assay, ibuprofen and mixed anhydrides of
ibuprofen with hexanoic and oleic acids (50 mg ibuprofen
equivalents) were dispersed in 1.0 mL of sterile water. Ac-
cording to the protocol, female Sprague–Dawley rats (250–
300 g, Harlan, Israel), six in each group were injected with
0.05 mL of 10% (w/v) aqueous suspension of yeast in the left
hindpaw followed by the injection of 100 ␮L of drug solution
and drug activity was determined by measuring the amount of
weight (in g) accumulated on the inflamed paw until the rat
withdraws its hindpaw under the weight probe (11). The
threshold of the rat to weight pressure was determined every
few hours for 24 h.
Scheme 1. Methods for the synthesis of mixed anhydrides.
action yields obtained when using ibuprofen acid chloride or
RESULTS
Ibuprofen mixed anhydrides based on binding ibuprofen ibuprofen acid when reacted with the corresponding acid or
to aliphatic and aromatic acids were prepared by four differ- acid chloride. The effect of the solvent on the reaction yield
ent methods as described in Scheme 1.
and the time to achieve maximal concentration of the mixed
The preferred method for the formation of mixed anhy- anhydride was examined (Fig. 1). Ibuprofen acid chloride-
drides with high purity and without further isolation or puri- :benzoic acid at a molar ratio 1:1.1 were reacted in the pres-
fication is the Schotten Baumann reaction of an activated ence of PVP at 0°C in different solvents and progress of the
carboxylic acid derivative with a carboxylic acid or the salt reaction was monitored by HPLC at 30, 90, and 270 min. As
thereof under mild conditions. shown in Fig. 1, the highest yield was obtained from the re-
In this method, ibuprofen acid chloride was reacted with action in chloroform for 30 min.
hexanoic acid or benzoic acid, and ibuprofen with hexanoyl
chloride or benzoyl chloride (Scheme 1a).
Mixed anhydrides of ibuprofen and fatty acids with the
chain length C3–C18 were prepared in chloroform for 30 min.
In each of these reactions, dichloromethane, petroleum All mixed anhydrides were liquids or semisolids at room tem-
ether, acetonitrile, chloroform, or toluene were used as sol- perature. The total anhydride yield was >85% and the mixed
vents. No significant differences in the mixed anhydride re- anhydride content is summarized in Table I.

208
Shaaya et al.
About 50% yield of mixed anhydrides were obtained as
determined by HPLC (Table I). Removal of a water molecule
from two carboxylic acids was used for mix anhydride forma-
tion (Scheme 1d). Ibuprofen was reacted with fatty acids us-
ing acetic anhydride as dehydrating agent to yield over 90%
of anhydrides with up to 50% mixed anhydride (Table I). The
mixed anhydrides were isolated by chromatography to yield
>90% pure compound.
Mixed Anhydrides Analysis
The formation of mixed anhydrides was determined by
HPLC as previously described (12). A simple reverse phase
HPLC was used for the quantitative analysis of symmetric
and mixed aliphatic and aromatic anhydrides. Anhydrides
and the corresponding acids were isolated using a C8 (octane
substituted silica) reverse phase column with a mixture of
acetonitrile:water (70:30 v/v) as mobile phase. No hydrolysis
of the anhydrides occurred during analysis.
Ibuprofen anhydrides were separated at retention times
of between 3 and 9 min using an isocratic flow of a 30:70 v/v
water:acetonitrile and UV detection at 254 nm. The retention
time was increased with the increase in chain lengths of fatty
acids. The detection limit for these anhydride derivatives was
set on 10 ␮g/mL. Ibuprofen acid and the fatty acids were
eluted at short retention times (2–5 min) and did not interfere
with the analysis of their corresponding anhydrides. For ex-
ample, the retention times for hexanoic anhydride, ibuprofen-
hexanoic anhydride, and ibuprofen anhydride were 3.9, 7.1,
and 7.7 min., respectively. This separation method was used
for the quantitative separation of mixtures of symmetric and
mixed anhydrides either by preparative HPLC or by flash
chromatography. The quantitative separation was conducted
at low temperature, preferably at refrigeration (4–8°C) to
avoid interchange of the mix anhydride to the corresponding
symmetric anhydrides.
Fig. 1. Effect of solvent on the formation of ibuprofen-benzoic acid-
mixed anhydrides. Ibuprofen acid chloride-benzoic acid at a molar
ratio 1:1.1 were reacted in the presence of poly(4-vinylpyridine) at
0°C in different solvents and progress of the reaction was monitored
by high-performance liquid chromatography at 30, 90, and 270 min.
The results obtained from the two-phase reaction
(Scheme 1b) are shown in Fig. 2a and b, which suggested that
the formation of mixed anhydride in the presence of PNO was
shorter (∼20 min) compared with the reaction time without
PNO. The mixed anhydride concentration was reduced dra-
matically over time in the presence of PNO followed by in-
creased concentration of the symmetric anhydride. Possible
reasons for these results are that the presence of PNO led to
the replacement of chloride with the acid, and anhydride re-
placement. Accordingly, the symmetric product was formed
with a higher concentration compared to the concentration
without PNO. Similar results were obtained when 18-crown-6
ether was used as phase transfer agent.
Mixed anhydrides were also prepared by anhydride in-
terchange (Scheme 1c). In this method, two symmetric anhy-
drides were reacted in solution or in bulk to form the corre-
sponding mixed anhydride.
IR analysis of aromatic and aliphatic anhydrides show
peaks at 1780 and 1730 cm−1 and at 1805 and 1740 cm−1
,
respectively. Mixed anhydrides of aromatic and aliphatic ac-
ids show peaks at 1800 and 1735 cm−1, which correspond to
the mixtures of aliphatic and aromatic anhydrides. An ab-
sorption peak at 1700 cm−1 indicated the existence of free acid
Table I. Ibuprofen-Fatty Acid Anhydrides Obtained from Different Methods
High-performance
liquid chromatography
reverse transcription (min)
Ibuprofen-fatty acid
anhydride (fatty residue)
Schotten
baumann
Anhydride-
interchange
Two-phase
Dehydration
CH3CH2
96
97
95
96
96
85
90
88
85
90
40
44
43
48
50
52
55
38
42
36
4.2
7.1
7.3
7.5
9.2
CH3(CH2)4
CH3(CH2)6
CH3(CH2)10
CH3(CH2)16
Note: Anhydrides were synthesized from the reaction between ibuprofen acid chloride and the fatty acids in chloroform for
30 min at 0°C using poly(4-vinylpyridine) as acid acceptor (Schotten Baumann); ibuprofen acid chloride and the fatty acids
reacted in water:chloroform 1:1 mixture using pyridine N-oxide PNO for 30 min at 0°C using poly(4-vinylpyridine (two
phase); symmetric anhydrides reacting in bulk at 60°C; ibuprofen and fatty acids reacted with acetic anhydride as dehydrating
agent. The total reaction yield was >90% and the mixed anhydride content were determined by high-performance liquid
chromatography.
a Structure of asymmetric ibuprofen anhydrides
.
b Mixed anhydride content was determined by HPLC, the retention times are given in minutes. Rest anhydride content was
symmetric anhydrides ibuprofen anhydride and fatty acid anhydride.
c Mixed anhydride yields of >90% were obtained after chromatography separation.

Anhydride Prodrugs for NSAIDs
209
The release rate of ibuprofen was a function of the fatty
acid chain length. Typically, 70% of the drug was constantly
released for 7 days from the ibuprofen-C3 derivative, whereas
only 30% was released from ibuprofen-C18 mixed anhydride
during the same time period. A comparison study of these
prodrugs at the pH 2 in physiological buffer was also per-
formed; however, no in vitro release could be monitored,
probably because the insolubility of the drug at acidic pH.
These in vitro drug release results are in correlation with the
results obtained for polyanhydride (14), in which, sebacic acid
and dodecanoic acid based polyanhydride had a slower hy-
drolysis rate in water than adipic acid based polyanhydride. In
both cases, the degradation rates were dependent on the
length of the alkanoic acid residue, which indicated that
longer alkanoic residue have slower degradation rates and
thus, anhydride prodrugs having longer alkanoic chains are
useful for extended release. In acidic pH, very little ibuprofen
was detected in the solution because of the insolubility of
ibuprofen in acidic solutions.
The mix anhydrides of ibuprofen were tested for their
analgesic activity using the Randall-Selito NSAID rat paw
edema model (11,12). The threshold of the inflamed rat paw
model to weight pressure was determined every few hours
for 24 h.
All ibuprofen mixed anhydrides produced significant an-
algesic effect in the yeast-inflamed paw test (Fig. 5). The long
chain derivative (oleic-ibuprofen mixed anhydride) was very
effective in increasing the pain threshold for 24 h, whereas the
shorter-chain derivative (hexanoic-ibuprofen mixed anhy-
dride) was effective for 24 h but at a lower threshold, and
intact ibuprofen was effective for less than 3 h at a compa-
rable dose. These data demonstrate the effectiveness of the
prodrugs for controlled delivery of drugs.
Fig. 2. Formation of ibuprofen-benzoate anhydride by the two phase
method with pyridine N-oxide (PNO; a) and without PNO (b). Con-
centrations of ibuprofen-benzoyic acid-mixed anhydride (᭹) and
benzoic acid symmetric anhydrides (᭿) obtained from the reaction of
ibuprofen sodium with benzoyl chloride in the presence of PNO (two-
phase reaction method). Ibuprofen sodium and benzoyl chloride (1.1:
1 molar ratio) reacted in water:dichloromethane 1:1 mixture at 0°C.
The concentration (in molar) of acids and anhydrides was determined
by HPLC.
products in the sample, which may be a result of hydrolysis or
unreacted acid. NMR spectra of mixed anhydride products of
ibuprofen and fatty acids conform with the structure of the
molecule. The H NMR spectrum of ibuprofen-hexanoate an-
hydride is given in Fig. 3, which show a good fit of the NMR
data to the molecule. All anhydride derivatives were either
liquids or semi solids.
DISCUSSION
Mixed anhydride derivatives of ibuprofen as model drug
with benzoic acid and aliphatic acids have been synthesized
for use as ibuprofen prodrugs with temporary hydrophobicity.
Mixed anhydrides were prepared by four methods with the
preferred method is the reaction between ibuprofen chloride
and the inert alkanoic acid in an organic solvent in the pres-
ence of insoluble poly(vinyl pyridine) as acid acceptor. Reac-
tion yields were in the range of 65–97%, with the mixed an-
hydride content greater than 85%.
The synthesis of symmetric anhydrides are well known
(15–17) and can be formed from two carboxylic acid mol-
ecules using a dehydrating agent. Common dehydrating
agents are acetic and propionic anhydrides, phosgene, diphos-
gene, dicyclohexylcarbodiimide, and methoxyacetylene.
These methods generally produce the symmetric anhydrides,
in addition to the desired mixed anhydrides. The preferred
method is the reaction between the carboxylic acid molecule
and an activated acid either is an organic solvent and an acid
acceptor, or in a mixture of organic and aqueous medium.
Examples of active acid derivative include acyl halides, or the
reaction product of a dehydrating agent with an acid or its
metal salt (15).
Stability of Mixed Anhydrides
Samples of ibuprofen-hexanoate mixed anhydride (90%
pure) were saved in sealed glass containers under dry argon at
–20, 4, 25, and at 37°C and the mix anhydride content was
followed for 7 days. Samples stored at refrigeration and –20°C
did not change the mixed anhydride content whereas the
samples stored at 25 or 37°C gradually decreased in mixed
anhydride content to a level of about 50% within 24 h, by an
anhydride interchange process to form the corresponding
symmetric anhydrides. These levels remain constant for the
rest of the experiment.
Release Behavior of Ibuprofen from Mixed Anhydrides
The hydrolytic degradation of ibuprofen-fatty acid mixed
anhydrides was studied by placing samples of mixed anhy-
drides in 0.1 M phosphate buffer pH7.4 at 37°C and monitor-
ing the concentration of ibuprofen in the buffer by HPLC.
The cumulative release (%) of ibuprofen from mixed anhy-
drides of ibuprofen with different fatty acids is shown in
Fig. 4.

210
Shaaya et al.
Fig. 3. 1H NMR spectrum of ibuprofen-hexanoate anhydride.
where M is H, or a metal ion such as Na, Ag, Tl, or Ca; X is amines (16). These acid acceptors form a salt with the hydro-
an halogen ion, or an active acid derivative; and R and RЈ are gen halide byproducts, which is mostly removed by filtration.
different organic residues of the carboxylic acid molecules.
However, the soluble amine salts remaining in the reaction
When MסH, hydrohalogenic acid is released during the mixture are difficult to remove. Thus, an alternative acid ac-
reaction that must be eliminated to avoid decomposition of ceptor used is a solid crosslinked PVP (16). This polymer
the anhydride product. The majority of acid acceptors used in binds to acid byproduct and can be isolated by filtration at the
the anhydride synthesis were pyridine and tertiary alkyl end of the reaction. Hence, we have used a solid amine as an
Fig. 4. In vitro release of ibuprofen from mixed anhydrides of ibu-
profen and fatty acids with different chain length as measured by Fig. 5. In vivo activity of ibuprofen mixed anhydrides as measured by
high-performance liquid chromatography. The measured mixed an- Randall-Selito test. The measured mixed anhydrides were as follows:
hydrides were: ibuprofen-C3 (᭿), ibuprofen-C6 (᭡), ibuprofen-C8
oleic-ibuprofen anhydride (᭿), hexanoic-ibuprofen anhydride (ࡗ),
(᭹), ibuprofen-C12 (ࡗ), ibuprofen-C18 (᭺), ibuprofen-C3 at pH 2 ( ). ibuprofen-ibuprofen anhydride (᭡), ibuprofen anhydride (᭹), and
*
Experiments were performed in phosphate buffer (pH 7.4), at 37°C. yeast ( ).
*

Anhydride Prodrugs for NSAIDs
211
acid acceptor. An additional problem associated with the ACKNOWLEDGMENTS
mixed anhydride synthesis in solution is the isolation of the
This work was supported in part by a grant from the
US-Israel Binational grant (BSF) and a grant from the Alex
Grass Center for Drug Design and Synthesis.
product from the symmetric and unreacted starting materials.
The most useful method is probably the reaction between the
acid or its metal salt and the acyl chloride of another acid.
The free carboxylic acid drug is conjugated to an inert
carrier or another drug via a hydrolytically labile anhydride
bond to alter the properties of the parent drug because the
prodrug properties also depend on the selected carrier. The
duration of drug release can be designed by altering the hy-
drophobicity of the mixed anhydride moiety from 1 to 24
methylenes. This modification will increase the hydrophobic-
ity of the prodrug, slow its hydrolysis, and increase the release
time of active parent drug. Hence, it is concluded that in
vivo/vitro hydrolysis of the anhydride conjugate is dependent
on the hydrophobicity of the anhydride groups. The main
limitation of this approach is the stability of the mixed anhy-
dride, which tend to form the corresponding symmetric an-
hydride during storage at ambient temperatures. It should be
noted that drugs containing reactive functional groups, such
as amino, hydroxyl or sulfhydryl, are less prominent to this
approach.
In this approach, the release rate of a free drug can be
manipulated by using different types of fatty acids. Thus, it
can be set at minimal (acidic pH) in the stomach while it can
be fast in the intestine, depending on the fatty acid derivative.
For systemic or localized delivery via intramuscular or sub-
cutaneous administration, a release of drug for periods of
weeks can be obtained with the only degradation byproduct
being a fatty acid. The results obtained suggest that anhydride
prodrugs can be a good alternate to overcome the problems
of carboxylic acid bearing drugs addressed in the introduction
part of this report. On one hand, anyone can protect the
carboxylic function can be temporarily masked by anhydride
bond to overcome stomach irritation. On the other hand, an-
hydride conjugated drugs with the acids having higher alkane
moiety provides higher absorption through lipid-water mem-
brane. In addition, this anhydride prodrug approach can be
applied for extended action/release of acidic drugs after ad-
ministration to the eye, skin, or oral intake.
REFERENCES
1. H. Bundgaard. Design of Prodrugs, Elsevier, Amsterdam, 1985.
2. I. T. Harrison, B. Lawis, P. Nelson, and W. Rooks. Nonsteroidal
antiinflammatory agents. I. 6-substituted 2-naphthylacetic acids.
J. Med. Chem. 13:203–205 (1970).
3. R. G. Child, A. C. Osterberg, A. E. Sloboda, and A. S. Tom-
cufcik. Fenbufen, a new anti-inflammatory analgesic: synthesis
and structure-activity relationships of analogs. J. Pharm. Sci. 66:
466–476 (1977).
4. L. B. Hansen, L. L. Christrup, and H. Bundgaard. Ketobemidone
prodrugs for buccal delivery. Acta Pharm. Nord. 3:77–82 (1991).
5. D. G. Waller and C. F. George. Prodrugs. Br. J. Clin. Pharmacol.
28:497–507 (1989).
6. R. M. Goetz and J. Holtz. Enhanced angiotensin-converting en-
zyme activity and impaired endothelium-dependent vasodilation
in aortae from hypertensive rats: evidence for a causal link. Clin.
Sci. 97:165–174 (1999).
7. F. Higashikawa, T. Murakami, T. Kaneda, A. Kato, and M. Ta-
kano. Dose-dependent intestinal and hepatic first-pass metabo-
lism of midazolam, a cytochrome P450 3A substrate with differ-
ently modulated enzyme activity in rats. J. Pharm. Pharmacol.
51:67–72 (1999).
8. N. Kumar, A.-C. Albertsson, U. Edlund, D. Teomim, A. Rasiel,
and A. J. Domb. Polyanhydrides. In A. Steinbuchel and Y. Doi
(eds.), Biopolymers: Polyesters (Part-B), Chap. 7, Wiley-VCH
Germany, 2002 pp. 203–234.
9. A. J. Domb, Z. H. Israel, O. Elmalak, D. Teomim, and A. Ben-
tolila. Preparation and characterization of carmustine loaded
polyanhydride wafers for treating brain tumors. Pharm. Res. 16:
762–765 (1999).
10. J. Tamada and R. Langer. The development of polyanhydrides
for drug delivery applications. J. Biomater. Sci. Polym. Ed. 3:315–
353 (1992).
11. M. A. Zahran, L. Kovacs, I. el-Sakka, E. B. Pedersen, and C.
Nielsen. The potential of aspirin in prodrug synthesis: a new po-
tential delivery system of AZT and FLT. Arch. Pharm. (Wein-
heim) 329:417–420 (1996).
12. R. J. Capetola, D. A. Shriver, and M. E. Rosenthale. Suprofen, a
new peripheral analgesic. J. Pharmacol. Exp. Ther. 214:16–23
(1980).
13. A. J. Domb. Quantitative analysis of mixed anhydrides. J. Chro-
matogr. 673:31–35 (1994).
14. A. J. Domb and R. Nudelman. In vivo and in vitro elimination of
aliphatic polyanhydrides. Biomaterials 16:319–323 (1995).
15. W. K. Fife and Z.-d. Zhang. Phase managed organic synthesis 3.
Symmetrical anhydrides from carboxylic acids via polymer as-
sisted reaction. Tetrahedron Lett. 27:4937–4940 (1986).
16. S. Patai. The Chemistry of Acid Derivatives-Part 1, John Wiley &
Sons, New York, 1979.
CONCLUSION
This work provides synthetic and characterization meth-
ods for mixed anhydrides of carboxylic acid-bearing drugs
and demonstrate the possible use of mixed anhydrides for
altering the physical properties of these drugs for improved
bioavailability.
17. C. A. Buehler and D. E. Pearson. Organic Synthesis (Vol. 2),
John Wiley & Sons, New York, 1974.