Anandamide prodrugs: 1. Water-soluble phosphate esters of arachidonylethanolamide and R-methanandamide

Article abstract of DOI:10.1016/S0928-0987(03)00044-7

Phosphate esters of arachidonylethanolamide (AEA) and R-methanandamide were synthesized and evaluated as water-soluble prodrugs. Various physicochemical properties (pK_a, partition coefficient, aqueous solubility) were determined for the synthesized phosphate esters. The chemical stability of phosphate esters was determined at pH 7.4. In vitro enzymatic hydrolysis rates were determined in 10% liver homogenate, and in a pure enzyme-containing (alkaline phosphatase) solution at pH 7.4. The intraocular pressure (IOP) lowering properties of R-methanandamide phosphate ester were tested on normotensive rabbits. The phosphate promoiety increased the aqueous solubility of the parent compounds by more than 16 500-fold at pH 7.4. Phosphate esters were stable in buffer solutions, but rapidly hydrolyzed to their parent compounds in alkaline phosphatase solution (t_1/2<<15 s) and liver homogenate (t_1/2=8-9 min). The phosphate ester of R-methanandamide reduced IOP in rabbits. These results indicate that the phosphate esters of AEA and R-methanandamide are useful water-soluble prodrugs.

Full text of DOI:10.1016/S0928-0987(03)00044-7

European Journal of Pharmaceutical Sciences 19 (2003) 37–43
www.elsevier.com/locate/ejps
Anandamide prodrugs
1
. Water-soluble phosphate esters of arachidonylethanolamide and
R-methanandamide
a ,
a
a
a
a
*
Juha Juntunen , Juhani Huuskonen , Krista Laine , Riku Niemi , Hannu Taipale ,
a
b
a
Tapio Nevalainen , David W. Pate , Tomi J a¨ rvinen
a
Department of Pharmaceutical Chemistry, University of Kuopio, PO Box 1627, FIN-70211, Kuopio, Finland
b
HortaPharm B.V., Amsterdam, The Netherlands
Received 30 October 2002; accepted 4 March 2003
Abstract
Phosphate esters of arachidonylethanolamide (AEA) and R-methanandamide were synthesized and evaluated as water-soluble prodrugs.
Various physicochemical properties (pK , partition coefficient, aqueous solubility) were determined for the synthesized phosphate esters.
a
The chemical stability of phosphate esters was determined at pH 7.4. In vitro enzymatic hydrolysis rates were determined in 10% liver
homogenate, and in a pure enzyme-containing (alkaline phosphatase) solution at pH 7.4. The intraocular pressure (IOP) lowering
properties of R-methanandamide phosphate ester were tested on normotensive rabbits. The phosphate promoiety increased the aqueous
solubility of the parent compounds by more than 16 500-fold at pH 7.4. Phosphate esters were stable in buffer solutions, but rapidly
hydrolyzed to their parent compounds in alkaline phosphatase solution (t1 / 2,,15 s) and liver homogenate (t1 / 258–9 min). The
phosphate ester of R-methanandamide reduced IOP in rabbits. These results indicate that the phosphate esters of AEA and
R-methanandamide are useful water-soluble prodrugs.

2003 Elsevier Science B.V. All rights reserved.
Keywords: Anandamide; Arachidonylethanolamide; Cannabinoid; Prodrugs; Water-soluble; Phosphate ester
1
. Introduction
inactivation by transport into cells (Beltramo et al., 1997),
followed by rapid metabolism. AEA is hydrolyzed to
arachidonic acid and ethanolamine by fatty acid amide
hydrolase (FAAH) (Cravatt et al., 1996). Abadji et al.
(1994) synthesized a chiral analog of AEA, R-methanan-
damide, which possessed improved stability and greater
Arachidonylethanolamide (AEA) was discovered in
992 by Devane et al. (1992), when they screened for
1
endogenous cannabinoid compounds. They tested the
ability of porcine brain extracts to displace a radiolabeled
cannabinoid probe ([ H] HU-243) in a ligand binding
3
affinity at the cannabinoid neuronal receptor (CB ). Im-
1
assay. After purification of promising brain extracts they
found a compound which they named anandamide, and
subsequently confirmed its structure by synthesis. AEA
was later shown to possess such cannabimimetic activities
as hypothermia, analgesia, catalepsy and hypomotility
proved stability and affinity of R-methanandamide is
caused by an R-methyl group adjacent to the amide
nitrogen. The recent findings have confirmed that the
endogenous cannabinoid system and anandamides have a
very important role in human physiology and that anan-
damides may be potential therapeutic agents in treatment
of several diseases (Pertwee, 1999, 2000).
As with the classical cannabinoids, anandamides are
very lipophilic and poorly water-soluble molecules. The
poor aqueous solubility of anandamides causes problems in
pharmacological experiments and decreases their pharma-
ceutical usefulness. Various approaches such as non-aque-
ous solvents (Wenger et al., 1995; Cabral et al., 1995) and
emulsifiers (Adams et al., 1995) have been used to
(
Crawley et al., 1993; Fride and Mechoulam, 1993; Smith
et al., 1994). Nowadays, the term anandamides is used to
describe structures that are similar to arachidonyl-
ethanolamide and its analogs.
In vivo effects of AEA have a short duration due to
*
Corresponding author. Tel.: 1358-17-16-2524; Fax:1358-17-16-
456.
E-mail address: juha.juntunen@uku.fi (J. Juntunen).
2
0
928-0987/03/$ – see front matter
 2003 Elsevier Science B.V. All rights reserved.
doi:10.1016/S0928-0987(03)00044-7

3
8
J. Juntunen et al. / European Journal of Pharmaceutical Sciences 19 (2003) 37–43
overcome this problem. Jarho et al. (1996) used cyclo-
dextrins to significantly improve both the aqueous solu-
bility and chemical stability of AEA.
The inlet capillary temperature was 225 8C. The samples
were dissolved in 50% methanol–water or in 100%
methanol (100 mg/ml) and 5 ml samples were injected.
The mobile phase consisted of acetonitrile–water mixture
(50:50) and the flow was set to 200 ml/min. Elemental
analyses were carried out on a ThermoQuest CE Instru-
ments EA 1110-CHNS-O elemental analyzer. High-per-
formance liquid chromatography (HPLC) determinations
were performed with a Merck LaChrom HPLC system
consisting of a model L-7250 programmable autosampler,
a model L-7100 intelligent pump, a model D-7000 inter-
face, a model L-7455 diode array detector and model
D-7000 HPLC system manager software (Hitachi, Tokyo,
Japan). A Purospher RP-18e (125 mm34.0 mm, 5 mm)
(Merck, Darmstadt, Germany) reversed-phase column was
Introduction of a polar or permanently charged moiety
to the anandamide structure opens up new possibilities for
use of these molecules as water-soluble prodrugs or
analogs. Phosphate esters have been synthesized in the past
to increase the water solubility of sparingly soluble
molecules (Cho et al., 1982; Varia et al., 1984; Jadhav et
al., 1996). Sheskin et al. (1997) synthesized AEA O-
phosphate and they speculated that it might be a precursor
of AEA in the body. They also measured binding affinities
of various arachidonyl amides, and AEA O-phosphate was
five times less active than AEA in their binding assay.
Hopper et al. (1996) introduced new phosphorylation
methods for mono- and polyunsaturated fatty acid chains,
including AEA. In the present study, we have synthesized
phosphate esters of AEA and R-methanandamide to evalu-
ate their use as water-soluble prodrugs for various dosage
forms and administration routes.
used for all HPLC separations. pH-metric pK determi-
a
nations were carried out using a Sirius PCA200 automatic
titrator (Sirius, Forest Row, UK) and data was analyzed
using Refine200 software (Sirius, Forest Row, UK).
2
2
.2. Chemistry
2
. Materials and methods
.2.1. General
Arachidonic acid (1) and AEA (2a) were purchased
2
.1. General procedures
from Deva Biotech (Hatboro, PA, USA). 2-Amino-1-pro-
panol was obtained from Fluka (Buchs, Switzerland) and
di-tert.-butyl diisopropylphosphoramidite from Aldrich
1
13
H NMR and C NMR spectra were recorded on a
Bruker Avance (Bruker, Rheinstetter, Germany) operating
at 500.1 MHz and 125.6 MHz, respectively. Chemical
shifts are reported as parts per million (d ) using TMS as an
internal standard. The following abbreviations are used for
(
Milwaukee, WI, USA). Tetrazole was purchased from
Acros Organics (Geel, Belgium). R-methanandamide (2b)
was synthesized (Scheme 1) using a slightly modified
method of Abadji et al. (1994). Phosphorylation of anan-
damides was carried out by a modified method of Hopper
et al. (1996).
1
describing H NMR data: s5singlet, d5doublet, t5triplet,
q5quartet, qui5quintet, m5multiplet and br5broad. Mass
spectra were recorded using a LCQ quadrupole ion trap
mass spectrometer (Finnigan, San Jose, CA). The spray
needle was set at 4.5 kV in the positive ion mode and to
2.2.2. Preparation of (5z,8z,11z,14z)-eicosa-5,8,11,14-
tetraenoic acid (2-hydroxy-1-methyl-ethyl)-amide (2b)
Arachidonic acid (1) (300 mg, 0.985 mmol) was
2
4.5 kV in the negative ion mode. The spray was
stabilized by a nitrogen sheath flow, the value set to 100.
Scheme 1. Synthesis of phosphate esters of AEA and R-methanandamide.

J. Juntunen et al. / European Journal of Pharmaceutical Sciences 19 (2003) 37–43
39
dissolved in dry methylene chloride (10 ml) and DMF (72
mg, 0.985 mmol) was added. The solution was cooled to
8C and oxalyl chloride (250 mg, 1.970 mmol) was added
dropwise. The solution was stirred at room temperature for
h. The solvent was evaporated to yield arachidonic acid
chloride. Arachidonic acid chloride was dissolved in dry
methylene chloride and the solution was cooled to 0 8C.
R(2)-2-amino-1-propanol (740 mg, 9.85 mmol) in 5 ml of
methylene chloride was added dropwise and the reaction
mixture was stirred for 1 h under a nitrogen atmosphere.
The mixture was washed twice with water (15 ml) and
dried with magnesium sulfate. The product was purified
with flash chromatography on 40 mm silica gel (J.T. Baker,
Deventer, Holland) using 4% methanol–96% methylene
2.2.4. Preparation of phosphoric acid di-tert.-butyl ester
2-((5z,8z,11z,14z)-eicosa-5,8,11,14-tetraenoylamino)-
propyl ester (3b)
0
Compound 3b was synthesized from R-methanandamide
(2b) (210 mg, 0.580 mmol), tetrazole (483 mg, 1.74
mmol) and di-tert.-butyl diisopropyl phosphoramidite (122
mg, 1.74 mmol) as described for 3a. The product was
purified by flash chromatography on silica gel with ethyl
acetate to give 150 mg (47%) of 3b as a clear oil. The
purification was done as fast as possible to avoid break-
2
1
down of the protecting tert.-butyl groups. H NMR
3
3
(CDCl ) d: 0.89 (t, J56.8 Hz, 3H), 1.21 (d, J56.8 Hz,
3
3H), 1.23–1.40 (m, 6H), 1.48 (s, 9H), 1.49 (s, 9H),
3
1.62–1.77 (m, 2H), 2.00–2.14 (m, 4H), 2.19 (t, J57.6 Hz,
chloride as eluent to give 279 mg (78.3%) of R-methanan-
2H), 2.73–2.87 (m, 6H), 3.84–3.93 (m, 1H), 3.95–4.04
1
damide (2b) as a slightly yellow oil. H NMR (CDCl ) d:
(m, 1H), 4.15–4.26 (m, 1H), 5.26–5.45 (m, 8H), 6.43 (s,
3
3
3
13
0
1
2
.84 (t, J56.8 Hz, 3H), 1.16 (d, J56.8 Hz, 3H), 1.23–
.39 (m, 6H), 1.63–1.76 (m, 2H), 2.05 (q, J56.9 Hz, 2H),
.12 (q, J56.8, 2H), 2.19 (t, J57.4 Hz, 2H), 2.76–2.88
1H). C NMR (CDCl ) d: 14.08, 17.15, 22.58, 25.56,
3
3
25.61, 25.62, 26.64, 26.70, 27.22, 29.33, 29.81, 29.84,
29.88, 31.52, 36.13, 45.15, 45.20, 69.66, 69.71, 127.56,
127.89, 128.21, 128.22, 128.59, 128.69, 129.13, 130.51,
172.79.
3
3
(
4
m, 6H), 3.48–3.56 (m, 1 H), 3.60–3.69 (m, 1H), 4.00–
1
3
.11 (m, 1H), 5.28–5.43 (m, 8H), 5.62 (br s, 1H).
C
NMR (CDCl ) d: 14.04, 17.04, 22.55, 25.45, 25.62, 25.63,
3
26.61, 27.20, 29.30, 31.50, 36.07, 47.80, 67.38, 127.51,
127.83, 128.14, 128.26, 128.61, 128.83, 129.03, 130.52,
173.64. ESI–MS: 362.3 (M11).
2.2.5. Preparation of phosphoric acid mono-[2-
((5z,8z,11z,14z)-eicosa-5,8,11,14-tetraenoyl amino)-ethyl]
ester (4a)
To a solution of 3a (95 mg, 0.175 mmol) in ethyl
acetate (2 ml) three drops of concentrated hydrochloric
acid were added. The mixture was stirred at room tempera-
ture for 6 h and evaporated in vacuo to give 75 mg (100%)
2
2
.2.3. Preparation of phosphoric acid di-tert.-butyl ester
-((5z,8z,11z,14z)-eicosa-5,8,11,14-tetraenoylamino)-ethyl
1
3
ester (3a)
of 4a. H NMR (CDCl ) d: 0.88 (t, J56.8 Hz, 3H),
3
AEA (2a) (222 mg, 0.638 mmol) and tetrazole (134 mg,
.92 mmol) were dissolved in a 50 ml mixture of dry
1.23–1.42 (m, 6H), 1.64–1.76 (m, 2H), 2.00–2.12 (m,
4H), 2.33 (t, J57.5 Hz, 2H), 2.70–2.86 (m, 6H), 3.51 (br
3
1
tetrahydrofuran and methylene chloride (1:1). Di-tert.-
butyl diisopropyl-phosphoramidite (531 mg, 1.92 mmol)
was added and the reaction mixture was stirred overnight
under a nitrogen atmosphere. After addition of 0.2 ml 30%
hydrogen peroxide, the reaction mixture was stirred for
s, 2H), 4.07 (br s, 2H), 5.25–5.43 (m, 8H), 8.43 (br s, 2H).
1
3
C NMR (CDCl ) d: 14.07, 22.58, 25.64, 25.65, 25.66,
3
26.62, 27.24, 29.34, 31.53, 35.49, 127.56, 127.86, 128.13,
128.40, 128.70, 128.83, 128.98, 130.55, 176.32. ESI–MS:
.
426.5 (M-1). Anal. (C H NO P 0.33H O) C, H, N.
2
2
38
5
2
2
h. The mixture was cooled to 0 8C and saturated sodium
metabisulfite solution was added to hydrolyze excess
hydrogen peroxide. The mixture was diluted with 50 ml of
ethyl acetate and washed twice with saturated sodium
metabisulfite solution (2350 ml). The organic phase was
dried with sodium sulfate and the solvent was evaporated
in vacuo. The product was purified by flash chromatog-
raphy on silica gel with ethyl acetate to give 190 mg
2.2.6. Preparation of phosphoric acid mono-[2-
((5z,8z,11z,14z)-eicosa-5,8,11,14-tetraenoyl amino)-
propyl] ester (4b)
Compound 4b was synthesized from 3b (110 mg, 0.199
1
mmol) as described for 4a to give 85 mg (97%) of 4b. H
3
3
NMR (CDCl ) d: 0.88 (t, J56.7 Hz, 3H), 1.20 (d, J56.0
3
Hz, 3H), 1.23–1.42 (m, 6H), 1.62–1.76 (m, 2H), 1.99–
2.14 (m, 4H), 2.22–2.38 (m, 2H), 2.73–2.88 (m, 6H), 3.91
(
55%) of 3a as a clear oil. The purification was done as
fast as possible to avoid breakdown of the protecting
(br s, 1H), 4.04 (br s, 1H), 4.21 (br s, 1H), 5.26–5.45 (m,
1
3
13
tert.-butyl groups. H NMR (CDCl ) d: 0.90 (t, J57.3 Hz,
8H), 8.25 (br s, 1H). C NMR (CDCl ) d: 14.09, 16.46,
3
3
3
3
H), 1.20–1.40 (m, 6H), 1.49 (s, 18H), 1.72 (qui, J57.6
22.58, 25.61, 25.64, 25.70, 26.55, 27.22, 29.33, 31.51,
35.56, 127.53, 127.85, 128.14, 128.33, 128.64, 128.84,
128.92, 130.52, 175.44. ESI–MS: 440.5 (M-1). Anal.
(C H NO P) C, H, N.
3
Hz, 2H), 2.01–2.14 (m, 4H), 2.22 (t, J57.6 Hz, 2H),
3
2
.74–2.87 (m, 6H), 3.53 (q, J55.1 Hz, 2H), 4.05 (qui,
3
13
J54.3 Hz, 2H), 5.29–5.43 (m, 8H), 6.52 (s, 1H).
C
2
3
40
5
NMR (CDCl ) d: 14.06, 22.58, 25.54, 25.66, 25.68, 26.76,
3
2
6
1
7.25, 29.34, 29.87, 29.90, 31.54, 36.07, 40.00, 40.04,
5.97, 66.02, 127.59, 127.92, 128.25, 128.63, 128.76,
29.14, 130.52, 173.23.
2.3. HPLC analysis
Gradient elution with a 20 mM phosphate buffer (pH

4
0
J. Juntunen et al. / European Journal of Pharmaceutical Sciences 19 (2003) 37–43
separated by centrifuging the tubes 10 min at 1700 rpm.
The concentration of phosphate esters in the buffer, before
and after the shaking, was determined by HPLC.
2
.7. Determination of pKa
pK was determined by the pH-metric technique (Av-
a
deef, 1993) using Sirius PCA200 computerized titration
system. Measurements were performed at 2561 8C under
an argon stream to minimize CO2 absorption. Sample
concentrations ranged from 0.40 mM to 0.65 mM. Two
independent measurements were made for each compound
and the results were combined into a multiset. HCl and
KOH solutions used in titrations were made using stan-
dardized ampoules (Merck, Darmstadt, Germany). Aver-
age ionic strength during the titrations was 0.151 M
(adjusted with 0.15 M KCl). Titrations were performed
from high to low pH (pH 9.88–1.66).
Fig. 1. HPLC separation of arachidonic acid, anandamides and their
phosphate esters.
7
.4)–90% acetonitrile solvent system achieved a good
chromatographic separation of arachidonic acid, anan-
damides and their phosphate esters (Fig. 1). A flow-rate of
0
.9 ml/min was used and compounds were detected at 211
nm.
2.8. Hydrolysis in enzyme solution
2
.4. Hydrolysis in aqueous solution
The rate of hydrolysis in alkaline phosphatase solution
was determined at 37 8C. Alkaline phosphatase (Type VII-
S: from bovine intestinal mucosa, 2.745 u/mg protein) was
purchased from Sigma (St Louis, MO, USA). 4a (0.5
mmol) or 4b (0.6 mmol) were each dissolved in an 185
mM borate buffer or 50 mM phosphate buffer (pH 7.4)
and each solution was filtered through a 0.45 mm mem-
brane filter. A tube containing each filtered solution (2 ml)
was placed in a 37 8C water bath and 4 ml (20.9 units) of
alkaline phosphatase was added. Samples (120 ml) were
removed from each tube and 120 ml of acetonitrile was
added to stop enzymatic hydrolysis. After centrifugation
(14 000 rpm, 10 min), samples were analyzed for the
remaining phosphate ester and for released anandamide by
HPLC.
The rates of chemical hydrolysis for phosphate esters at
7 8C were determined in 50 mM phosphate buffer (pH
.4, ionic strength 0.5) and in 185 mM borate buffer (pH
.4). The solutions were prepared by dissolving an appro-
3
7
7
priate amount of phosphate esters in buffer solution (initial
concentration 198–246 mM). The filtered solutions (Milli-
pore 0.45 mm) were placed in a water bath at 37 8C and
samples were withdrawn at appropriate intervals. Samples
were analyzed for remaining phosphate ester by HPLC.
2
.5. Aqueous solubility
The aqueous solubilities of phosphate esters were de-
termined at room temperature in 185 mM borate buffer
pH 7.4). Compounds 4a (8.43 mg) and 4b (3.81 mg)
(
were added to 1 ml of buffer solution and the solution was
vigorously shaken for 4 h, filtered (0.45 mm Millipore),
diluted and analyzed for phosphate ester by HPLC. The pH
of the mixtures was held constant throughout the study.
2.9. Hydrolysis in liver homogenate
The hydrolysis of 4a and 4b was studied in 10% rabbit
liver homogenate at 37 8C. The 20% liver homogenate was
prepared by homogenizing rabbit liver with four equiva-
lents of isotonic phosphate buffer (pH 7.4). The homoge-
nate was centrifuged for 90 min at 90003g at 4 8C and the
supernatant was stored at 280 8C until used. One volume
of 1.15 mM phosphate ester solution in borate buffer (pH
7.4) was added to one volume of stirred, pre-incubated
(37 8C) 20% liver homogenate to give 10% liver homoge-
nate. The solution was kept at 37 8C and 200 ml samples
were withdrawn and added to 300 ml of cold acetonitrile.
After mixing and centrifugation (14 000 rpm, 10 min), the
samples were analyzed for the remaining phosphate ester,
and for released anandamide, by HPLC.
2
.6. Distribution coefficient
The distribution coefficients (log D) of phosphate esters
a and 4b were determined at 25 8C in a 1-octanol-borate
4
buffer (pH 7.4 and pH 5.0) system. Before use, the
-octanol was saturated with borate buffer by vigorously
1
shaking for 24 h. A known amount of phosphate ester was
dissolved in the borate buffer and the pH was checked and
adjusted (if necessary). The solution was filtered using a
0
.45 mm membrane filter and 2 ml of the filtered solution
was shaken with 0.4 ml 1-octanol for 4 h. The phases were

J. Juntunen et al. / European Journal of Pharmaceutical Sciences 19 (2003) 37–43
41
2
2
.10. IOP studies
in differences of the means was tested using the Fisher’s
Protected Least Significant Difference (PLSD) method at
the 95% confidence level.
.10.1. Formulation of eyedrops
R-methanandamide was dissolved in aqueous 15% HP-
b-CD solution. Earlier studies (Pate et al., 1995, 1998)
have demonstrated that topical administration of HP-b-CD
3. Results and discussion
(up to 30% solution) does not affect the IOP of rabbits.
The pH of R-methanandamide solution was adjusted to 7.4
with sodium hydroxide and the solution was made isotonic
with sodium chloride. Isotonicity of the solution was
confirmed by using an Osmostat OM-6020 Auto-Osmome-
ter (Kyoto Daiichi Kagaku, Kyoto). The phosphate ester of
R-methanandamide (4b) was dissolved to an isotonic 50
mM phosphate buffer at pH 7.4. Final drug concentration
of R-methanandamide and of the phosphate ester of R-
methanandamide in the eyedrop solutions was 6.91 mmol/
ml. The isotonic 50 mM phosphate buffer was used as a
negative control treatment. Drug concentrations were
analyzed by HPLC.
3.1. Hydrolysis in aqueous solution
The effect of a buffer system on the stability of
phosphate esters 4a and 4b was considerable. Phosphate
esters 4a and 4b hydrolyzed more slowly in 185 mM
borate buffer (t1 / 2511.9 days and 13.9 days, respectively)
compared to 50 mM phosphate buffer (t1 / 252.4 days and
3.1 days, respectively), which is most probably due to a
catalytic effect of phosphate ions on the hydrolysis of the
phosphate ester group (Table 1).
3.2. Aqueous solubility
2
.10.2. Intraocular pressure (IOP) measurements
The method for IOP measurements has been described
Jarho et al. (1996) measured the aqueous solubility of
2
6
AEA to be 0.4 mg/ml (1.15310
M) at pH 7.4.
earlier (Pate et al., 1998; Laine et al., 2002). The ex-
perimental animals used were normotensive Dutch Belted
rabbits of either gender (2.4–3.5 kg, n55). The rabbits
were housed singly in cages under standard laboratory
conditions: 12 h dark/12 h light cycle, 20.060.5 8C and
Phosphate esters 4a and 4b were completely dissolved
during the solubility experiment. Because there was no
undissolved compound left after the experiment, exact
solubilities can not be given. The maximum solubility of
4a and 4b could not be measured due to the small amount
of each phosphate ester available and their high respective
aqueous solubilities. The aqueous solubilities of 4a and 4b
in a pH 7.4 solution at room temperature were determined
5
5–75% relative humidity. Rabbits were given water and
food ad libitum except during the tests. Animals were
treated in accordance with the ARVO Statement for the use
of animals in ophthalmic and vision research.
2
3
to be at least 8.15 mg/ml (19.06310 M) and 3.8 mg/ml
2
3
During the tests, rabbits were placed in plastic restrain-
ing boxes located in a quiet room. A drop (25 ml) of the
test solution was instilled unilaterally into each left eye of
the rabbits. During installation, the upper eyelid was pulled
away slightly from the globe. IOP was measured using a
BioRad (Cambridge, MA) Digilab Modular One
Pneumatonometer. Before each measurement, one or two
drops of 0.06% oxybuprocaine were applied to the cornea
to eliminate discomfort. For each determination, at least
two readings were taken from the treated and untreated
eye, and the mean of these readings was used. IOP of the
rabbits were measured 1 and 0 h before, and at 0.5, 1, 2, 3,
(8.60310
M), respectively (Table 2). In any case,
introduction of a phosphate moiety to the anandamide
structure significantly increased the aqueous solubility of
the parent drug (Table 2).
3.3. Distribution coefficient
Compound 4b showed a higher distribution coefficient
(log D) at pH 5.0 and 7.4 compared to 4a (Table 2) which
is probably due to the a-methyl substituent on the former
structure. The high partition coefficients suggest ion-pair
partitioning. Partition of 4a or 4b between octanol and the
aqueous phase is strongly dependent on the pH of the
aqueous phase due to their respective ionizable phosphate
moieties. The lipophilic character of both 4a and 4b
4
and 5 h after the administration of the eyedrop. IOP at
the time of eyedrop administration (0 h) was used as a
baseline value. Baseline IOPs ranged between 25.5–16.8
mmHg. All studies were set up using a randomized
crossover design. At least 72 h of wash-out time was
allowed for the rabbits between dosings.
Table 1
Half-lives (days) of 4a and 4b in phosphate buffer (50 mM; pH 7.4) and
in borate buffer (185 mM; pH 7.4) at 37 8C
2
.10.3. Statistical analysis of IOP data
Compound
t1 / 2 (days)
T1 / 2 (days)
Results are given as a change in IOP (mmHg)
Phosphate buffer
Borate buffer
(185 mM; pH 7.4)
mean6S.E.M. A one-factor analysis of the variance
ANOVA) for repeated measurements was used to test the
(
50 mM; pH 7.4)
(
4
4
a
b
2.4
3.1
11.9
13.9
statistical differences between the R-methanandamide, the
phosphate ester 4b and control treated groups. Significance

4
2
J. Juntunen et al. / European Journal of Pharmaceutical Sciences 19 (2003) 37–43
Table 2
Aqueous solubility (one determination), distribution coefficient at pH 5.0 and 7.4 (log D, mean6S.D.; n53) and pK of AEA, 4a and 4b
a
Compound
Solubility
mM)
pH 7.4)
Solubility
compared
to AEA
Log D5.0
(pH 5.0)
Log D7.4
(pH 7.4)
pKa1
pKa2
(
(
2
3a
AEA
1.15310
.19.06
.8.6
–
–
–
–
2.68
2.65
–
6.88
6.73
4
4
a
b
.16500
.7500
1.9160.02
2.6060.30
1.1560.09
1.5360.02
a
Jarho et al., 1996.
decreases when pH is increased. These phosphate esters 4a
and 4b have high aqueous solubilities (.8.15 mg/ml and
which is a good phosphoryl acceptor (Simopoulos and
Jencks, 1994).
.
3.8 mg/ml, respectively) and adequate distribution co-
efficients (log D7.451.15 and 1.53, respectively) which is a
3.6. Hydrolysis in liver homogenate
very good combination for further drug development.
The breakdown of 4a and 4b in liver homogenate
released AEA and R-methanandamide, respectively. Both
3
.4. Determination of pka
4
a and 4b were hydrolyzed with half-lives of 8 and 9 min,
respectively, (Table 3) and AEA was further hydrolyzed to
arachidonic acid. The breakdown of R-methanandamide to
arachidonic acid was probably prevented by the R-a-
methyl group.
The two pK s of phosphate ester 4a and 4b are shown in
a
Table 2. Goodness-of-fit values (Avdeef, 1993) for the
multiset of two determinations were 1.48 and 1.31 for 4a
and 4b, respectively. The good solubility of the phosphate
esters at pH 7.4 can be readily explained by the pKa-
values. At pH 7.4 the hydroxyl group with the lower pKa
3
.7. Intraocular pressure (IOP) measurements
(
2.65) is completely ionized and more than 80% of the
Both R-methanandamide (dissolved in HP-b-CD solu-
other hydroxyl group is in ionized form.
tion) and phosphate ester 4b caused a statistically signifi-
cant (P,0.05) fall of IOP in the treated eye when
compared to a buffer solution (Fig. 2). The maximal
observed decrease of IOP was 3.160.7 mmHg and
2.460.4 mmHg at 2 h after administration of 173 nmol of
R-methanandamide or 4b, respectively. No statistically
significant difference between R-methanandamide and 4b
could be observed.
3
.5. Hydrolysis in enzyme solution
Enzymatic hydrolysis of phosphate esters 4a and 4b in
borate buffer was very rapid (completely hydrolyzed
before first sample) in the presence of alkaline phosphatase
(
Table 3), releasing AEA and R-methanandamide, respec-
tively. The buffer system is important in these enzymatic
hydrolyses, as the reaction was much slower in phosphate
buffer than in borate buffer. Both phosphate and borate are
known to be inhibitors of alkaline phosphatase (Fernandez
et al., 1981). Phosphate ion inhibits alkaline phosphate
much more effectively than borate ion, which is logical as
the inorganic phosphate binds to alkaline phosphatase
thereby inhibiting the enzyme. Inorganic phosphate is also
produced when alkaline phosphatase hydrolyses phosphate
esters. The slow dissociation of inorganic phosphate from
the enzyme could be avoided by using buffer like Tris,
Administration of 173 mmol of R-methanandamide or
4b did not cause statistically significant decrease of IOP in
the corresponding untreated eyes when compared to a
buffer solution (data not shown) which suggests that the
reduction of IOP is a local effect.
Table 3
Half-lives (min) (mean6S.D., n53) of 4a and 4b in alkaline phosphatase
solution and liver homogenate at 37 8C
Compound
t1 / 2 (min)
t1 / 2 (min)
Alkaline phosphatase
10% Liver
homogenate
Borate
buffer
Phosphate
buffer
Fig. 2. IOP changes (mean6S.E.M., n55) in treated eyes of normoten-
sive rabbits after unilateral ocular administration of isotonic phosphate
buffer (d), 173 mmol R-methanandamide (n) or 173 nmol phosphate
ester 4b (s). Symbols (*) indicate data significantly different from values
for the phosphate buffer.
a
4
4
a
b
Rapid hydrolysis
Rapid hydrolysis
20969
473624
861
960
a
a
Completely hydrolyzed before the first sample (15 s).

J. Juntunen et al. / European Journal of Pharmaceutical Sciences 19 (2003) 37–43
43
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4
. Conclusions
Phosphate esters of AEA and R-methanandamide sig-
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The authors would like to thank the Academy of Finland
and the National Technology Agency (Finland) for finan-
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