A peptide prodrug approach for improving bisphosphonate oral absorption

Article abstract of DOI:10.1021/jm980645y

This work was aimed at improving the absorption of bisphosphonates by targeting carrier systems in the intestine and the intestinal peptide carrier system (hPEPT1), in particular. ¹⁴C-Labeled pamidronate and alendronate as well as radiolabeled

Full text of DOI:10.1021/jm980645y

J . Med. Chem. 2000, 43, 3641-3652
3641
A P ep tid e P r od r u g Ap p r oa ch for Im p r ovin g Bisp h osp h on a te Or a l Absor p tion
Aviva Ezra,† Amnon Hoffman,† Eli Breuer,‡ Ivan S. Alferiev,‡ J ukka Mo¨nkko¨nen,# Naama El Hanany-Rozen,‡
Gal Weiss,‡ David Stepensky,† Irith Gati,† Hagit Cohen,† Soili To¨rma¨lehto,# Gordon L. Amidon,§ and
Gershon Golomb*,†
Departments of Pharmaceutics and Medicinal Chemistry, School of Pharmacy, Faculty of Medicine,
The Hebrew University of J erusalem, P.O. Box 12065, J erusalem 91120, Israel, Department of Pharmaceutics,
University of Kuopio, Kuopio, Finland, and College of Pharmacy, University of Michigan, Ann Arbor, Michigan
Received J une 21, 1999
This work was aimed at improving the absorption of bisphosphonates by targeting carrier
systems in the intestine and the intestinal peptide carrier system (hPEPT1), in particular.
14C-Labeled pamidronate and alendronate as well as radiolabeled and “cold” peptidyl-
bisphosphonates, Pro-[3H]Phe-[14C]pamidronate, and Pro-[3H]Phe-[14C]alendronate were syn-
thesized. In situ single-pass perfusion studies revealed competitive inhibition of transport by
Pro-Phe, suggesting peptide carrier-mediated transport. Prodrug transport in the Caco-2 cell
line was significantly better than that of the parent drugs, and the prodrugs exhibited high
affinity to the intestinal tissue. Oral administration of the dipeptidyl prodrugs resulted in a
3-fold increase in drug absorption following oral administration in rats, and the bioavailability
of Pro-Phe-alendronate was 3.3 (FTIBIA) and 1.9 (FURINE) times higher than that of the parent
drug. The results indicate that the oral absorption of bisphosphonates can be improved by
peptidyl prodrugs via the hPEPT1; however, other transporters may also be involved.
In tr od u ction
A number of geminal bisphosphonates have been
approved for clinical use in Paget’s disease, tumor
osteolysis, and hypercalcemia of malignancy, and alen-
dronate has been recently approved for osteoporosis1-5
(see Figure 1). The major disadvantage of the clinically
utilized bisphosphonates is their poor absorption from
the GI tract. Less than 1% of the oral dose is absorbed,
and absorption is suppressed in the presence of food.6-10
Furthermore, the absorption is variable between indi-
viduals and within the same individual, making ac-
curate oral dosage of these compounds difficult. The poor
absorption of bisphosphonates, tetraacids almost com-
pletely ionized at physiological pH, is attributed to their
high polarity which prevents transcellular transport
across the epithelial barriers.11 In addition, the very
poor solubility of the metal salts/complexes formed from
bisphosphonates in the GI tract is another possible
reason for poor absorption.12 In the treatment of os-
teoporosis the variable and less than 1% absorption of
the bisphosphonates is clinically effective, since it is
administered chronically and most of the absorbed drug
strongly binds to bone. However, to obtain an effective
antiresorptive effect in tumor osteolysis, a relatively
high dose of bisphosphonates must be given parenter-
ally.13 The preferred mode of administration is by iv
infusion, since im and sc injections are associated with
local necrosis. Thus, if increased absorption could be
attained, oral administration would be a viable delivery
F igu r e 1. Structures of pamidronate, alendronate, Pro-Phe-
pamidronate, and Pro-Phe-alendronate.
* To whom correspondence should be addressed. Phone: 972-2-
6757504. Fax: 972-2-6757246. E-mail: golomb@cc.huji.ac.il.
† Department of Pharmaceutics, The Hebrew University of J erusa-
lem.
system in situations where higher doses are required.
In addition bisphosphonate therapy in osteoporosis
would improve significantly.
A relatively novel method to increase membrane
permeability is by utilizing the carrier systems of the
brush-border membrane of intestinal mucosa.14 The
‡ Department of Medicinal Chemistry, The Hebrew University of
J erusalem.
#
University of Kuopio.
§ University of Michigan.
10.1021/jm980645y CCC: $19.00 © 2000 American Chemical Society
Published on Web 09/13/2000

3642 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20
Ezra et al.
Ta ble 1. Distribution of [14C]Alendronate and
active transporter, hPEPT1, is responsible for di/tri-
peptide absorption and for absorption of â-lactam an-
tibiotics,15 renin inhibitors,16 and angiotensin-converting
enzyme (ACE) inhibitors.17,18 It was found that dipeptide
prodrugs Phe-R-methyldopa, R-methyldopa-Phe, and
R-methyldopa-Pro demonstrate more than 10 times
higher intestinal permeability than the parent drug L-R-
methyldopa, a polar amino acid, that is poorly trans-
ported by the amino acid transporter.19 There are no
reports on orally absorbable bisphosphonates or on
application of the peptide prodrug approach to enhance
GI absorption of these drugs. In this work bisphospho-
nates (pamidronate and alendronate, the most active
bisphosphonates approved for clinical use) were con-
verted into the peptidyl prodrugs prolyl-phenylalanyl-
pamidronate (Pro-Phe-pamidronate) and prolyl-phen-
ylalanyl-alendronate (Pro-Phe-alendronate). This work
was aimed at improving the absorption of bisphospho-
nates by targeting carrier systems in the intestine and
the intestinal peptide carrier system, in particular. It
was hypothesized that the prodrug would be actively
transported into the cytosol and would subsequently be
hydrolyzed by cystosolic enzymes to the parent drug.
Pro-[3H]Phe-[14C]alendronate in Rat (n ) 6) Tissues after iv
Dose of 1 mg/kg Alendronate
distribution (% dpm or % dpm/g tissue)
tissue
[
14C]alendronate
Pro-Phe-[14C]alendronate
urine
tibia
femur
kidney
liver
19.4 ( 6.9
5.2 ( 0.4
23.9 ( 5.01
4.2 ( 0.45*
3.2 ( 0.28
1.59 ( 0.38*
0.119 ( 0.02*
2.2 ( 1.41
3.8 ( 0.6
0.28 ( 0.1
0.048 ( 0.02
0.88 ( 0.3
feces
*Differences were termed statistically significant by two-tailed
nonparametric Mann-Whitney U-test (n ) 6, mean ( SD, p <
0.05).
dronate, respectively) and significantly lower amounts
of alendronate were disposed in the tibia following
prodrug administration (1.2 times lower). Analysis of
3
the H isotope in urine and bone specimens resulted in
similar results (data not shown).
3. Or a l Absor p tion a n d Bioa va ila bility.
3.1. P r o-P h e-p a m id r on a te: Following oral admin-
istration of 20 mg/kg Pro-[3H]Phe-[14C]pamidronate
(equivalent to 10 mg/kg pamidronate) the urine amount
of 14C (bisphosphonate moiety) was 3 times higher than
that found following pamidronate administration (0.64
and 0.21% dpm, respectively, Figure 2). The levels of
14C in the tibia, resulting from the administration of
prodrug, were 4.8 times higher than those found fol-
lowing parent drug administration (Figure 2). The
detection of the prodrug labeled with the two isotopes
in the urine revealed 2.5 times higher amounts of the
peptide moiety (3H counts) than that of the bisphospho-
nate (14C counts, Figure 3). In soft tissues, especially
in the liver, high concentrations of the peptide moiety
were detected, with similar concentrations in the tibia
(Figure 3).
Resu lts
1. Syn th esis of Bisp h osp h on a tes a n d P r od r u gs.
Pamidronate-1-14C and alendronate-1-14C were synthe-
sized from 3-aminopropanoic-1-14C acid and 4-aminobu-
tyric-1-14C acid, respectively, by modification of a pro-
cedure involving the reaction of carboxylic acids with
phosphorus trichloride, in the presence of pyridine
hydrochloride.20
N-t-Boc-L-prolyl-L-phenylalanine was activated by
conversion to N-hydroxysuccinimide ester, which in turn
was condensed with the aminoalkyl-bisphosphonates in
the presence of 3.5 equiv of diisopropylethylamine. This
reaction was carried out in aqueous 2-propanol. In this
solvent mixture it was possible to obtain a homogeneous
reaction mixture containing both the ionic bisphospho-
nate salt and the “organic” peptide derivative. It was
possible to monitor the progress of the reaction by 31P
NMR spectroscopy, due to the small shift of the 31P
signal caused by the attachment of the peptide chain.
The peptidyl-bisphosphonates were isolated from the
reaction mixtures by precipitation with benzathine and
recovery using a cation exchange column. Pro-Phe-
pamidronate was characterized by elemental analysis,
1H and 31P NMR spectroscopy, and FAB mass spec-
trometry. Pro-Phe-alendronate was characterized by
elemental analysis, 1H and 31P NMR spectroscopy, and
FAB mass spectrometry. The labeled bisphosphonates
and peptidyl-bisphosphonates were identified only by
1H and 31P NMR spectroscopy.
2. iv Ad m in istr a tion . The distribution of Pro-Phe-
alendronate and alendronate in calcified and noncalci-
fied tissues of rats is shown in Table 1. Following iv
administration of 2 mg/kg Pro-[3H]Phe-[14C]alendronte
(equivalent to 1 mg/kg alendronate) the amount of 14C
(bisphosphonate moiety) in urine was not significantly
different than that found following alendronate admin-
istration (Table 1). Significantly higher amounts of
alendronate were disposed in soft tissues of liver and
kidney following prodrug administration (5.6 and 2.5
times higher in the liver and kidney, prodrug and alen-
3.2. P r o-P h e-a len d r on a te: Following oral adminis-
tration of 20 mg/kg Pro-[3H]Phe-[14C]alendronte (equiva-
lent to 10 mg/kg alendronate) the urine amount of 14C
(bisphosphonate moiety) was 2 times higher than that
found following 10 mg/kg alendronate administration
(0.74 and 0.32% dpm, respectively, Figure 2). Most of
the prodrug was found in the feces (79.4%, Figure 2).
Following prodrug administration, the concentration of
14C in the tibia was 2.7 times higher than that found
following parent drug administration (Figure 2). The
analyses of the different isotopes (3H and 14C) in the
urine revealed higher levels of the peptide moiety (3H
counts) than the bisphosphonate moiety (14C counts),
(3.83 and 0.74% dpm, respectively, Figure 3). The levels
3
of H found in the feces were lower than those of 14C
(Figure 3). The levels of the peptide moiety (3H) in the
soft tissues were 73 times higher than those of the
bisphosphonate moiety (14C) (e.g. in the liver, 0.22 and
0.003% dpm/g, respectively) with similar levels in the
tibia (Figure 3).
3.3. Bioa va ila bility: The bioavailability of Pro-Phe-
alendronate calculated according to drug accumulation
in bone and in urine, FTIBIA and FURINE, were 1.74% and
3.10%, respectively (Table 2). The bioavailabilities of
the parent drug, FTIBIA and FURINE, were, respectively,
3.3 and 1.9 times lower than that of the prodrug
(Table 2). Similar results were found for pamidronate
(Table 2).

Improving Bisphosphonate Oral Absorption
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20 3643
F igu r e 2. Disposition of Pro-[3H]Phe-[14C]pamidronate, [14C]pamidronate, Pro-[3H]Phe-[14C]alendronate, and [14C]alendronate
(14C counts) in rat urine, feces, and tibia 24 h after po administration. The tested compounds (10 and 20 mg/kg pamidronate/alen-
dronate and Pro-Phe-pamidronate/alendronate, respectively) were administered po to rats housed in metabolic cages, and the
amounts of 14C were determined 24 h after administration. Data are expressed as % dpm in feces and urine and as % dpm/g tibia
weight. *Differences were termed statistically significant by the Mann-Whitney one-tailed test (mean ( SE, n ) 6, p < 0.05).
4. Mech a n ism of P r od r u g Absor p tion .
cm/s, respectively; for Pro-Phe alendronate 1.10 × 10-5
and 2.3 × 10-5 cm/s, respectively). The transport rate
of Pro-Phe-pamidronate was suppressed 2-fold by the
competitive inhibitor Pro-Phe.
4.1. Ga str ic a bsor p tion : To determine the site of
prodrug absorption and the mechanism, prodrug ab-
sorption was studied in pylorus-ligated rats. The amount
of pamidronate and Pro-Phe-pamidronate detected in
the tibia after peroral administration in pylorus-ligated
rats is presented in Figure 4. The levels of 14C in bone
following oral administration of pamidronate and Pro-
Phe-pamidronate in pylorus-ligated rats were similar
in both the femur (9.36 × 10-3 and 1.17 × 10-2% dpm/
g, respectively, data not shown) and the tibia (9.37 ×
10-3 and 1.08 × 10-2% dpm/g, respectively). However,
a markedly better absorption of the prodrug was ob-
served in control animals (approximately 3.8-fold in the
femur and 4.7-fold in the tibia).
4.3. Up ta k e of P r o-P h e-a len d r on a te in th e in tes-
tin a l w a ll: Figure 6 shows the time-dependent uptake
of the prodrug in comparison to alendronate in rat
intestine wall tissue after oral administration. The
uptake vs time was in a bell shape, and the amount of
prodrug in the intestinal wall was higher at all time
3
points than that of the parent drug (14C and H counts).
These results suggest that the prodrug has higher
affinity to the intestinal tissue than the parent drug.
This may suggest a carrier/active transport, although
other reasons such as hydrophobic bonding are possible.
5. Tr a n sp or t in th e Ca co-2 Cell Lin e. Table 3
shows the apical (ap) to basolateral (bl) and bl to ap
fluxes of mannitol, Pro-Phe, alendronate, pamidronate,
and their dipeptidyl prodrugs across Caco-2 cell mono-
layers. The prodrugs Pro-Phe-pamidronate and Pro-Phe-
alendronate (bisphosphonate moiety, 14C) from the ap
to bl direction were transported significantly better
than the parent drugs (2.2 and 3.8 times higher,
respectively).
4.2. In situ sin gle-p a ss p er fu sion : Figure 5 shows
the effective permeability coefficients of the parent
drugs, the dipeptide, and the prodrugs. The perme-
ability of Pro-Phe-pamidronate was studied in the
absence and presence of a competitive inhibitor (Pro-
Phe). Compared to the parent drugs, the effective
permeability coefficients of Pro-Phe-pamidronate and
Pro-Phe-alendronate were 7.9- and 6.9-fold higher,
respectively. No difference was found between the
values of Peff calculated on the basis of 14C or H counts
Analyses of the different isotopes (3H and 14C) re-
vealed that the transport of the dipeptidyl moiety (Pro-
3
(for Pro-Phe-pamidronate 1.79 × 10-5 and 1.90 × 10-5

3644 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20
Ezra et al.
3
F igu r e 3. Disposition of Pro-[3H]Phe-[14C]pamidronate and Pro-[3H]Phe-[14C]alendronate (14C and H counts) in rat urine, feces,
tibia, and liver organs 24 h after po administration. The tested compounds (10 and 20 mg/kg pamidronate/alendronate and Pro-
Phe-pamidronate/alendronate, respectively) were administered po to rats housed in metabolic cages, and the amounts of 14C and
3H were determined 24 h after administration by means of a sample oxidizer. Data are presented as % dpm in feces and urine
and as % dpm/g tibia or liver weight. *Differences were termed statistically significant by the Mann-Whitney one-tailed test
(mean ( SE, n ) 6, p < 0.05).
Ta ble 2. Prodrug Oral Bioavailabilitya
direction in comparison to the bl to ap direction. These
Pro-Phe-[14C]-
alendronate alendronate pamidronate pamidronate
Pro-Phe-[14C]-
results may suggest active/carrier-mediated transport.
6. Biocon ver sion . The most significant hydrolysis
of Pro-Phe-pamidronate was found in the intestinal
mucosal homogenate of the jejunum segment (36%,
Table 4). When the prodrug concentration was increased
from 0.25 to 1 mM, the hydrolysis in the same concen-
tration of jejunum segment was decreased to 12%. No
significant hydrolysis was detected in artificial gastric
juice or other segments of the GI tract (Table 4).
7. Biologica l Activity.
FURINE
FTIBIA
3.10*
1.74*
1.65
0.52
3.17*
1.65*
1.74
0.44
a
Bioavailability values of the prodrugs were estimated by the
14
ratios of 14Cpo
/ C accumulated in bone and urine 24 h following
iv
administration (see Experimental Section). *Significantly different
from the respective parent drug by one-tailed nonparametric
Mann-Whitney U-test (n ) 6, mean ( SD, p < 0.05).
Phe) was significantly higher from the ap to bl direction
in comparison to the bl to ap direction, indicating active
transport. The transport of the bisphosphonate moieties
(14C counts of pamidronate and alendronate) of both
prodrugs was insignificantly higher from the ap to bl
7.1. An tica lcifica tion effects: Table 5 presents the
in vitro effects of pamidronate and Pro-Phe-pamidronate
on the extent of HAP precipitation. Both drugs signifi-
cantly inhibited HAP formation at both concentrations

Improving Bisphosphonate Oral Absorption
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20 3645
F igu r e 4. Disposition of Pro-[3H]Phe-[14C]pamidronate in rat
tibia 2 h after po administration in pylorus-ligated rats and
24 h after po administration in control animals. [14C]Pamidr-
onate and Pro-[3H]Phe-[14C]pamidronate (10 mg/kg pamidr-
onate) were administered po to pylorus-ligated rats to avoid
intestinal absorption (active carrier-associated absorption).
The amounts of 14C and 3H were determined 2 h after po
administration. Data are presented as % dpm (14C counts)/g
tibia weight. *Differences were termed statistically significant
by the Mann-Whitney one-tailed test (mean ( SE, n ) 6, p
< 0.05).
F igu r e 5. Effective permeability coefficients of rat jejunum
for the parent drugs (pamidronate and alendronate), the
dipeptide (Pro-Phe), and the prodrugs (Pro-Phe-pamidronate/
alendronate). The jejunal segment was perfused by single pass
in situ (rate of 0.19 mL/min and 0.09 mM tested compound).
The permeability of Pro-Phe-pamidronate was tested in the
absence and presence of a competitive inhibitor (4 mM Pro-
Phe). *Differences were termed statistically significant by the
Mann-Whitney one-tailed test (mean ( SE, 4 e n e 8, p <
0.05), except for Pro-Phe-pamidronate vs Pro-Phe-pamidronate
+ Pro-Phe, p ) 0.058.
(0.1 and 1 mM). The in vitro anticalcification activity
of pamidronate and Pro-Phe-pamidronate was found to
decrease at the higher concentration. This apparent
inactivity was due to the low solubility of the calcium
salts/complexes of the former two compounds, rather
than increased HAP formation.21
Figure 7 depicts the inhibition of bioprosthetic heart
valve tissue calcification by Pro-Phe-pamidronate, Pro-
Phe-alendronate, pamidronate, and alendronate. The
prodrugs and bisphosphonates significantly inhibited
tissue calcification in the following order: alendronate
) pamidronate > Pro-Phe-alendronate ) Pro-Phe-
pamidronate. The anticalcification effect of the various
compounds was not associated with adverse effects on
somatic growth as evidenced by the normal weight gain
of the treated animals.
prodrugs. Unfortunately, the former approach has led
to damage of the epithelial cells and/or has resulted in
only very limited success when applied to bisphospho-
nates.12,22 A recent paper describes a prodrug of clodr-
onate (clodronic acid dianydrides), but no absorption/
permeation studies have been reported.23 Because of its
involvement in the oral absorption of â-lactam antibiot-
ics, renin inhibitors, and ACE inhibitors, the oligopep-
tide transporter has received much attention as a drug
delivery system. Hu et al.19 studied the possibility to
improve the absorption of L-R-methyldopa by synthesiz-
ing peptide analogues that target to the peptide carrier
system. The permeability of the dipeptide prodrugs Phe-
R-methyldopa, R-methyldopa-Phe, and R-methyldopa-
Pro increases significantly in comparison to the parent
compound. Recently similar results were found for
valcyclovir in comparison to acyclovir.24 In this study
we applied the same approach to bisphosphonates. The
brush-border membrane of intestinal mucosa contains
an active transporter for di- and tripeptides (hPEPT1).
In this work, the bisphosphonates were coupled to the
dipeptide, proline-phenylalanine, which is known to be
actively transported by the peptide carrier.25 Earlier
studies in animals performed by Lin et al.8,26 have
demonstrated that following systemic administration,
7.2. Effects on HAP d issolu tion : Table 5 shows the
effect of pamidronate and Pro-Phe-pamidronate on the
dissolution of HAP tablets at pH 5. Pro-Phe-pamidr-
onate was found to be as effective as pamidronate in
inhibiting HAP dissolution in this in vitro model.
Discu ssion
There are two common ways to improve oral absorp-
tion of highly hydrophilic polar drugs: (1) the use of
absorption enhancers and (2) the design of lipophilic

3646 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20
Ezra et al.
Ta ble 5. Inhibition of HAP Formation in 3.87 mM Calcium
and 2.32 mM Phosphate Metastable Solution by Pamidronate
and Pro-Phe-pamidronate and Inhibition of HAP Dissolution in
Vitro (at steady state) by Adsorbed Pamidronate and
Pro-Phe-pamidronate
inhibition of
HAP dissolution,
% of control
inhibition of
HAP formation,
% of initial concentration
compound
NaCl
0.24 (mM)
0.048 (mM)
1.0 (mM)
0.1 (mM)
100.0 ( 8.7 100.0 ( 3.7 18.0 ( 0.5
55.7 ( 2.6* 79.4 ( 4.9* 46.9 ( 1.8* 63.8 ( 1.9*
17.0 ( 2.1
pamdronate
Pro-Phe-Pam 59.1 ( 3.7* 79.5 ( 5.2* 40.5 ( 0.71* 83.8 ( 1.26*
a
The amount of calcium dissolved in the NaCl group was
termed as 100%. *Differences were termed statistically significant
by the Kruskal-Wallis and Dunn’s tests (4 e n e 10, mean (
SD, p < 0.05).
F igu r e 6. Time-dependent uptake of Pro-Phe-alendronate
and alendronate by the intestine following po administration.
The tested compounds (10 and 20 mg/kg alendronate and Pro-
Phe-alendronate, respectively) were administered po to rats
3
housed in metabolic cages, and the amounts of 14C and H were
determined 0.25, 0.5, 1.5, and 3 h after administration by
means of a sample oxidizer. Data are presented as % dpm/g
intestine (duodenum) weight (mean ( SE, n ) 2/time point).
Ta ble 3. Apical to Basolateral and Basolateral to Apical
Fluxes of Mannitol, Proline-Phenylalanine, Alendronate,
Pamidronate, and Their Dipeptidyl Prodrugs
(Pro-[3H]Phe-[14C]alendronate and
Pro-[3H]Phe-[14C]pamidronate) across Caco-2 Cell Monolayers
at 37 °Ca
permeability ×10-7 (cm/s)
compound
ap-bl
bl-ap
mannitol
Pro-Phe
alendronate
Pro-Phe-alendronate (Pro-Phe)
Pro-Phe-alendronate (alendronate)
pamidronate
Pro-Phe-pamidronate (Pro-Phe)
Pro-Phe-pamidronate (pamidronate)
2.6 ( 1.0
22.9 ( 8.5
0.6 ( 0.1
10.3 ( 2.0**
2.3 ( 2.4**
0.5 ( 0.2
5.0 ( 0.2**
1.1 ( 0.5**
2.2 ( 1.0
11.4 ( 0.6*
0.6 ( 0.1
5.5 ( 1.2*
1.6 ( 0.7
0.4 ( 0.2
4.1 ( 0.2*
0.9 ( 0.06
a
Differences were termed statistically significant by the Mann-
Whitney one-tailed test (mean ( SD, n > 6, p < 0.05), except for
Pro-Phe-alendronate (14C counts, alendronate) vs alendronate, p
) 0.0564. *Significantly different in comparison to ap-bl (p <
0.05). **Significantly different in comparison to parent bisphos-
phonate (p < 0.05).
F igu r e 7. Effect of Pro-Phe-pamidronate and Pro-Phe-alen-
dronate delivered by miniosmotic pumps on bioprosthetic heart
valve tissue calcification implanted sc in rats for 14 days. Mini
osmotic pumps containing 0.014 M drug solution, delivering
test compounds at 1.68 µmol/kg/day, were co-implanted sc with
bioprosthetic heart valve tissue (1 × 1 cm). Data are presented
as µg calcium/mg dry tissue weight. *Differences were termed
statistically significant by the Kruskal-Wallis and Dunn’s
two-tailed test (mean ( SE, n ) 6, p < 0.05).
Ta ble 4. Enzymatic Hydrolysis of Pro-Phe-pamidronate in Rat
J ejunum Homogenate and Artificial Gastric J uicea
GI segment
duodenum
prodrug concn (mM)
hydrolysis (%)
1
5
jejunum
1
12
0.5
0.25
0.5
0.5
0.25
20
36
6
ileum
artificial gastric juice
(with pepsin)
no hydrolysis
no hydrolysis
pharmacokinetics of bisphosphonates have been char-
acterized to a limited extent in humans. It is difficult
to quantify the concentration of a bisphosphonate in
biological fluids, especially in plasma, following thera-
peutic doses of alendronate due to the rapid clearance
and limited analytical methods. Since alendronate and
pamidronate are largely deposited in bone where they
have a long half-life, their absorption is estimated in
animals by measuring the drug concentration in bone
tissues.27 In addition, since renal excretion is the only
a
The extent of hydrolysis was determined by radioactive
measurements of 3H derivatives (represented by the fragments
cleaved off from pamidronate, Pro-Phe, and/or Phe) after selective
precipitation of calcium-bisphosphonate complex (n ) 2, 16 h).
alendronate was rapidly cleared from plasma and either
taken up by bone (60-70%) or excreted by the kidney
(30-40%). In addition, the half-life of alendronate in
bone is very long, more than 200 days in rats. The

Improving Bisphosphonate Oral Absorption
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20 3647
route of elimination of the drugs, absorption can also
be assessed by measuring the drug concentration in the
urine.27 The results of this study show that the oral
administration of the dipeptidyl prodrugs Pro-Phe-
pamidronate and Pro-Phe-alendronate resulted in a
significant increase of drug absorption. Drug concentra-
tion in the urine, following oral administration of the
prodrugs Pro-Phe-pamidronate and Pro-Phe-alendr-
onate, was between 2 and 3 times higher than that
obtained following administration of the parent drugs
(Figure 2). Similarly, the concentration of pamidronate
in the tibia from the prodrug was higher than that
following oral administration of the parent drug (Figure
2). Alendronate concentration in the tibia following oral
administration of Pro-Phe-alendronate revealed the
same phenomenon with drug concentration from the
prodrugs higher than that following parent drug ad-
ministration (Figure 2). The bioavailability of Pro-Phe-
alendronate was 3.3 (FTIBIA) and 1.9 (FURINE) times
higher than that of alendronate. The iv experiment with
Pro-Phe-alendronate compared to alendronate revealed
2.5 (bisphosphonate moiety) and 3.4 (peptide moiety)
times higher levels in the feces (not quite significant, p
) 0.0931). The latter finding indicates that there might
be excretion of the drug via bile. This finding, coupled
with the relatively high levels of the prodrug in soft
tissues (kidney and liver) even 24 h post-prodrug
administration, as well as the small extent of prodrug
reconversion noted after iv administration, suggests
that the disposition of the intact prodrug is different
from that of the parent drug. Thus the bioavailability
calculated according to drug accumulation in bone and
urine is not precise, and the actual bioavailability is
probably higher.
primarily via the dipeptide transporter system and
drugs that are absorbed by passive diffusion in the
Caco-2 cell line. The conclusion of this study was that
the in vitro permeability through Caco-2 cells is not
quantitatively predictive of in vivo absorption for pep-
tide-like drugs absorbed via the dipeptide transporter
system. Despite this limitation of the Caco-2 system,
transport of the prodrugs (dipeptidyl moiety and bis-
phosphonate moiety) from the ap to bl direction was
found to be significantly higher than that of the parent
drug. The ap to bl and bl to ap transports of alendronate
and pamidronate were similar, which indicates that the
transport of the bisphosphonates is passive. The dipep-
tidyl moiety was transported significantly higher from
the ap to bl direction in comparison to the bl to ap
direction. The bisphosphonate moiety had an insignifi-
cantly better transport from the ap to bl direction.
(Table 3). The prodrug’s high affinity to the intestinal
tissue and the increased transport in Caco-2 cells
further support a carrier-mediated transport of the
peptidyl prodrugs.
Since it is known that the peptide carrier system has
broad specificity and excellent capability, the relatively
small increment of prodrug absorption (2-4 times) may
suggest that the prodrug inhibits transport. In addition,
the absorption was inhibited by an hPEPT1 competitive
inhibitor transport supporting the peptidyl carrier
mechanism. However, it is plausible to assume that the
increased absorption of the prodrugs could be attributed
to another active carrier system as well.
The contribution of the hydrolyzed free form should
also be considered. Since the levels of 14C (bisphospho-
nate moiety) were higher than those of 3H (peptide
moiety) in the feces (Figure 3), the free form of the drug
hydrolyzed in the lumen could not contribute to the
prodrug absorption. On the other hand, free drug
discharged at the intestinal wall by brush-border en-
zymes could contribute in the prodrug increased absorp-
tion. This is supported by the high affinity of the
prodrugs to the intestinal tissue (Figure 6). The free
drug at the brush-border will not chelate with calcium
or other heavy metals and, therefore, could contribute
to the transport.
Although proline and phenylalanine are unpolar
(hydrophobic) amino acids, the octanol/buffer partition
coefficient of the prodrugs was similar to that of the
parent drugs (0.001, log P ) -3). Since the prodrug has
a similar lipophilicity and 2 times larger molecular
weight than the parent drug, the increased absorption
of the prodrugs could not be ascribed to increased
passive diffusion. The mechanism of the improved
absorption of the prodrugs was elucidated by the per-
fusion and pylorus-ligated rat studies. Previous studies
on the absorption of Gly-Gly and Gly-Leu across the
gastrointestinal tract in human showed no transport in
the stomach but did show the following order of trans-
port activity in the small intestine: jejunum > ileum >
duodenum where the active carrier system exists.28 In
situ single-pass perfusion studies showed that the
intestinal absorption of Pro-Phe-pamidronate was re-
duced by Pro-Phe (Figure 5). The competitive inhibition
by the dipeptide suggests that Pro-Phe-pamidronate
shares a common carrier with the small peptides,
hPEPT1. In addition, the extent of absorption of our
prodrug from the stomach was significantly less than
that observed from the GI tract (Figure 4). Moreover,
pamidronate absorption from the stomach was similar
to that observed after po administration in control
animals (Figure 4). This is further indirect evidence
supporting the hypothesis that the increased absorption
of the prodrug is due to active transport.
To be useful as an oral prodrug of bisphosphonates,
the peptidyl prodrugs have to withstand enzymatic
attacks in the GI tract prior to absorption. The stability
of Pro-Phe-pamidronate was studied in artificial gastric
juice and in homogenates derived from three regions of
the intestine. In the experimental time span, Pro-Phe-
pamidronate was stable in artificial gastric juice, sug-
gesting that the prodrug is resistant to pepsin and low
pH and might be administered as a plain tablet with
no need for entero-coating. As shown in Table 4, Pro-
Phe-pamidronate underwent relatively little degrada-
tion in the intestinal cell homogenate. The decreased
hydrolysis of Pro-Phe-pamidronate in the intestinal
mucosal homogenate of the jejunum as the concentra-
tion increased may suggest a saturation of the enzyme.
The metabolism of the prodrugs in the body fluids/
tissues can also be estimated from the analytical results
of the different isotopes after oral administration. As
expected, the concentrations of the amino acids in the
urine and soft tissues, especially in the liver (a metabolic
organ), were substantially higher than those of the
Chong et al.29 investigated whether there is a quan-
titative difference between drugs that are absorbed

3648 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20
Ezra et al.
(0.39 g, 3.76 mmol having total radioactivity 1 mCi), water
(0.30 mL, 16.4 mmol) and pyridine (1.08 mL, 13.4 mmol) to
yield 0.682 g (72%) of product, 31P NMR (solvent: D2O + Na2-
CO3, pH ) 5) δ ) 18.0 ppm, t, J ) 12 Hz.
bisphosphonates (Figure 3). The high concentrations of
the amino acids in the feces (Figure 3) suggest relatively
little bioconversion of the prodrugs in the intestinal
lumen, thus indicating that the absorption of the
prodrugs was achieved with no significant hydrolysis.
The results presented indicate that relatively high
concentrations of the peptidyl prodrug may not com-
pletely convert to the parent drug after systemic uptake.
Therefore examination of the biological activities of the
prodrugs takes on added importance. For this purpose
two experimental models that are commonly used for
predicting and evaluating the anticalcification effects
of bisphosphonates were used. In the in vitro model Pro-
Phe-pamidronate was found to be as effective as pam-
idronate in inhibiting HAP formation (Table 5). Good
inhibition of HAP formation in vitro implies high
activity as a crystal poison in vivo.30,31 Indeed, the
prodrugs significantly inhibited the calcification of
tissues implanted subdermally in rats. The mechanism
of action of bisphosphonates in inhibiting bioprosthetic
heart valve tissue calcification is mainly attributed to
a direct physicochemical effect on crystal growth and
dissolution (“crystal poison”).32 It can be assumed that
the greater the affinity of the bisphosphonate to the
crystal, the greater its “crystal poison” effect.
To evaluate the potential antiresorptive properties of
the bisphosphonate prodrugs, we examined the effects
on inhibition of HAP dissolution at pH 5. In the
experimental conditions employed, using a HAP tablet
and two concentrations of Pro-Phe-pamidronate, it was
possible to detect small differences between the activi-
ties of the prodrug in comparison to the parent drug.
The prodrug significantly inhibited HAP dissolution
(Table 5). To some extent, the in vivo antiresorption
effects of the compounds can be predicted on the basis
of the results obtained by this model.33 Classical prodrug
is inactive in the parent form; therefore, the prodrug
activity results and the low metabolism in vivo suggest
that the derivatives may not be behaving as classical
prodrugs.
L-P r olyl-L-p h en yla la n ylp a m id r on a t e (P r o-P h e-p a m -
idr on ate). N,N′-Dicyclohexylcarbodiimide (520 mg, 2.52 mmol)
was added to a stirred and ice cooled solution of N-tert-
butoxycarbonyl-L-prolyl-L-phenylalanine (Boc-L-Pro-L-PheOH)
(660 mg, 1.82 mmol) and N-hydroxysuccinimide (HOSU, 230
mg, 2 mmol) in dichloromethane (CH2Cl2, 12 mL). After
stirring for 30 min at 0 °C the solution was further stirred for
48 h at room temperature. The precipitated dicyclohexylurea
was filtered and washed with CH2Cl2 (10 mL). The combined
organic solvents were evaporated, and the residue was treated
with pentane (25 mL). The crystalline product was filtered and
washed with pentane to give 803 mg N-tert-butoxycarbonyl-
L-prolyl-L-phenylalanine N-hydroxysuccinimide ester (Boc-Pro-
Phe-OSU). This compound was added to a solution of 3-amino-
1-hydroxypropane 1,1-bisphosphonic acid (277 mg, 1.18 mmol)
and diisopropylethylamine (0.71 mL, 4.15 mmol) in 1.7 mL
2-propanol and 0.25 mL water. After stirring the solution for
48 h, the solvent was evaporated to afford a syrup which was
taken up in water (13 mL) and acetic acid (0.53 mL) resulting
in a solution of pH 3. The solution was filtered to remove
impurities and evaporated. A 14% solution of benzathine
diacetate in water (13 mL), was added to the residue causing
the formation of a viscous syrup. After 2 h the supernatant
liquid was removed by decantation and a new portion of 13
mL benzathine solution was added. The mixture was then left
overnight. The supernatant was decanted again, and the
viscous syrup was washed with water (3 × 5 mL) and ether (2
× 12 mL). The washing solutions were removed by decanta-
tion. The syrup was dissolved in a solution of water-methanol
(1:2, 80 mL) and passed through a column of Dowex 50 (H+
form, 30 mL). The column was then washed with water-
methanol (1:2, 80 mL) until the pH of the eluted liquid was
4-5. The eluted solution was evaporated to give a mixture of
N-tert-butoxycarbonyl-L-prolyl-L-phenylalanylpamidronate and
l-prolyl-L-phenylalanylpamidronate. To this mixture 1.8 mL
of trifluoroacetic acid was added to complete the removal of
the tert-butoxycarbonyl protecting group. After 30 min at room
temperature the reaction mixture was evaporated to dryness
followed by coevaporation with methanol (5 × 25 mL). The
solid obtained was filtered and washed with methanol to yield
388 mg (70%) of L-prolyl-L-phenylalanylpamidronate. NMR (in
D2O with Na2CO3, pH ) 7): 31P{H} 17.9 ppm; 1H 1.4-2.4 (m,
6H, Pro 2â CH2, pamidronate â CH2); 3-3.5 (m, 6H, Pro R
CH2, pamidronate γ CH2, Phe benzyl CH2); 4.25 (m, 1H, Pro
R CH); 4.52 (m, 1H, Phe R CH); 7.2-7.4 (m, 5H, Phe aromatic).
MW calcd 479.36, found by FAB MS 479.8. Anal. (C17H27N3O9P2‚
In conclusion, it was shown in this work that the
bioavailability of bisphosphonates can be enhanced by
using the peptidyl prodrug approach. The increased oral
absorption of the prodrugs was mediated by an active
carrier-mediated transport.
2H2O) C; H; N. [R]20 ) -21.9° (c ) 2, in 1% triethylamine in
D
methanol).
L-P r olyl-L-p h en yla la n yla len d r on a te (P r o-P h e-a len d r -
on a te). This compound was synthesized using the method
described for the synthesis of L-Prolyl-L-phenylalanylpamidr-
onate by adding alendronate (2 g, 8.03 mmol) to a solution of
Boc-Pro-Phe-OSU (5.141 g, 11.2 mmol) and diisopropyleth-
ylamine (4.2 mL, 28.6 mmol) in 11.4 mL 2-propanol and 1.8
mL water. After stirring the solution for 48 h, the reaction
was worked up as previously described to yield 2.09 g (53%)
of L-prolyl-L-phenylalanylalendronate. NMR (in D2O with
Exp er im en ta l Section
3-Am in o-1-h yd r oxyp r op a n e 1,1-Bisp h osp h on ic-1-14
C
Acid (P am idr on ate-1-14C). 3-Aminopropanoic-1-14C acid (0.374
g, 4.2 mmol having total activity 1 mCi), water (0.30 mL, 16.4
mmol) and pyridine (1.08 mL, 13.4 mmol) were placed in a
flask provided with an efficient condenser and a powerful
mechanical stirrer. Dry hydrogen chloride gas was introduced
into the mixture with vigorous stirring until saturation. After
cooling to 40 °C, phosphorus trichloride (1.25 mL, 14.3 mmol)
was added, and the mixture was stirred on a bath gradually
heated from room temperature to 95 °C, and then kept at 95
°C for 1 h. Water (0.3 mL) and concentrated hydrochloric acid
(0.2 mL) were added, and the mixture was hydrolyzed by
heating for 5 h at 135 °C (in the bath). Addition of water (1.5
mL) and 2-propanol (2.0 mL) to the reaction mixture caused
crystallization to give 0.765 g (77%) of radioactive pamidr-
onate, pure according to 31P NMR (solvent: water + 10% of
pyridine) δ ) 17.5 ppm, t, J ) 12.5 Hz.
1
Na2CO3, pH ) 7): 31P δ ) 19.5 ppm; t, (J ) 12 Hz); H 1.7-
1.97 (m, 8H, Pro 2â CH2, alendronate â + γ CH2); 3.02-3.05
(m, 4H, pamidronate γ CH2, Phe benzyl CH2); 3.1-3.2 (2H,
m, Pro R CH2) 4.1-4.15 (m, 1H, Pro R CH) 4.2-4.25 (m, 1H,
Phe R CH); 7.2-7.4 (m, 5H, Phe aromatic Hs). MW calcd
493.39, found by FAB MS 493.2. Anal. Calcd (C18H29N3O9P2‚
2H2O) C, 40.84; H, 6.28; N, 7.94. Found C, 41.14; H, 6.12; N,
6.6. [R]20D ) -24.45°(c ) 2, in 1% triethylamine in methanol).
L-P r olyl-L-p h en yla la n yl-3H -p a m id r on a t e-1-14
C (P r o-
P h e-3H-p a m id r on a te-1-14C). A reaction mixture containing
L-phenylalanine-1-14C (496 mg, 3.0 mmol, total activity 5 mCi)
water (1 mL), diisopropylethylamine (0.5 mL), 2-propanol (20
mL), water (2 mL) and N-tert-butoxycarbonylprolyl-N-hydroxy-
4-Am in o-1-h ydr oxybu tan e 1,1-Bisph osph on ic-1-14C Acid
(Alen d r on a te-1-14C). This synthesis was carried out similarly
to the previous experiment using 4-aminobutyric-1-14C acid

Improving Bisphosphonate Oral Absorption
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20 3649
succinimide ester (844 mg, 2.7 mmol) was stirred at ambient
temperature for 24 h. The mixture was then filtered and
washed with 2-propanol. The filtrate was transferred to a
separatory funnel and extracted with dilute phosphoric acid
and then with water. After drying, the ethereal solution was
evaporated to yield 722 mg (74%) Boc-Pro-PheOH-3H. To the
dipeptide dissolved in CH2Cl2 and cooled in an ice bath,
N-hydroxysuccinimide (252 mg, 2.19.mmol) and dicyclohexy-
lcarbodiimide (569 mg, mmol 2.76 mmol) were added, and the
mixture was allowed to stand at ambient temperature for 48
h. After removing the dicyclohexylurea by filtration, the
solution was evaporated, the residue was taken up in pentane,
and the precipitate was collected to yield Boc-Pro-Phe-OSU-
3H. To the activated dipeptide a solution of pamidronate-1-
14C (480 mg, 2.0 mmol), diisopropylethylamine (1.21. ml), water
(0.42 mL) and 2-propanol (2.84 mL) was added, and the
solution was stirred at ambient temperature for 48 h. The
reaction was worked up as described previously for Pro-Phe-
pamidronate to yield 482 mg (50%) of L-prolyl-L-phenylala-
nylpamidronate, having 1H and 31P NMR spectra identical
with those of the “cold” product.
L-P r olyl-L-ph en ylalan yl-3H-alen dr on ate-1-14C (P r o-P h e-
3H-a len d r on a te-1-14C). To a solution of labeled activated
dipeptide Boc-Pro-Phe- OSU-3H (918 mg, 2.0 mmol) in
CH2Cl2, alendronate-1-14C (449 mg, 1.8 mmol) in a solution
(1.21 mL), consisting of 2-propanol (2.56 mL) and water (0.4
mL) and diisopropylethylamine (1.1 mL, 6.42 mmol) was
added. The solution was left to be stirred at ambient temper-
ature for 48 h. The reaction mixture was worked up as
described previously to yield 294 mg (30%) L-prolyl-L-pheny-
lalanylalendronate having a 31P{H} NMR spectrum identical
with that of the cold product.
Phe-bisphosphonate and/or Phe-bisphosphonate and/or bis-
phosphonate on one hand, to Pro-Phe and/or Phe on the other.
Bioavailability values of the prodrugs were estimated by the
ratios of 14Cpo/14Civ accumulated in bone or urine 24 h following
administration corrected for the dose:8
FTIBIA ) (amount tibiapo × Div)/(amount tibiaiv × Dpo) × 100
FURINE ) (amount urinepo × Div)/(amount urineiv × Dpo) × 100
In a separate experiment the time dependent distribution
of Pro-Phe-alendronate and alendronate after oral administra-
tion was examined. The distribution of the prodrug and the
parent drug in the intestinal tissue was determined after po
administration of 10 mg/kg bisphosphonate. Male Sabra rats
weighing 250-300 g were kept in metabolic cages for 1 week
prior to the investigation for acclimatization, with free access
to water and food. Each group of rats (n ) 10) received an
oral dose of the tested compound by a stomach tube after an
overnight fast. The animals (2 rats/time point) were eutha-
nized and the intestinal tissues were excised at 0.25, 0.5, 1.5,
3 and 24 h. Radioactivity levels in intestine (duodenum) tissue
3
were determined by combustion to 14CO2 and H2O by means
of a sample oxidizer (Sample Oxidizer, Packard, Meriden, CT)
as described above.
Ga str ic Absor p tion . The active carrier system is present
only in the intestine. To determine site of prodrug absorption,
the absorption of pamidronate and Pro-Phe-pamidronate from
the stomach was studied in rats following pylorus ligation. The
peritoneal cavity was opened by midline incision under ket-
amine and xylazine anesthesia; the pylorus was ligated; the
incision was sutured; and the rats were allowed to recover for
iv Ad m in istr a tion . The tested compounds and dosages
were [14C]pamidronate (1 mg/kg, 1µCi/mg), Pro-[3H]Phe-[14C]-
pamidronate (1 mg/kg of pamidronate, 14C 0.5 µCi/mg; 3H 2.6
µCi/mg), [14C]alendronate (1 mg/kg, 14C 1 µCi/mg) and Pro-
[3H]Phe-[14C]alendronate (1 mg/kg of alendronate, 14C 0.5 µCi/
mg; 3H 2.5 µCi/mg). Male Sabra rats weighing 220-250 g were
kept in metabolic cages for 1 week prior to the investigation
for acclimatization, with free access to water and food. An
indwelling cannula was inserted into the rat’s right jugular
vein under light ether anesthesia 1 day before drug adminis-
tration. A dose of 1 mg/kg of the tested compound was
dissolved in iso-osmotic vehicle (0.3 mL saline). The rats were
euthanized by overexposure to ether 24 h after drug admin-
istration, and the tibia, femur, kidney, liver, intestine, spleen,
urine and feces were obtained and frozen until assay. Radio-
activity levels in tissues were determined by combustion to
14CO2 and 3H2O by means of a sample oxidizer (Sample
Oxidizer, Packard, Meriden, CT) and liquid scintillation count-
ing, using appropriate standard curves (enabling separate
a
period of 2 h. Pamidronate (10 mg/kg) and Pro-Phe-
pamidronate (20 mg/kg, equivalent to 10 mg/kg of pamidr-
onate) were administered orally by stomach tube to 2 rats in
each group. Two hours following drug administration the rats
were euthanized. The tibia, femur, kidney, liver and spleen
were then obtained, and the amounts of the drug and the
prodrug were determined as described above.
In Situ Sin gle-P a ss P er fu sion . The technique was similar
to previous reports.34 The rats (male Sabra rats weighing 200-
250 g) were fasted overnight (12-18 h) before each experiment.
Anesthesia was induced by im injection of nembutal (0.1 mL/
100 g). The rats were kept on a heating slide warmer to
maintain their body temperature. The abdominal cavity was
opened by a middling incision of 3-4 cm. The jejunum was
located and cannulated at 2-4 cm below the ligament of Treitz
and about 10 cm distal to the first incision.34 The tubing
between the peristaltic pump and the inlet cannulae was
placed in a water bath of 37 °C. After cannulation, the
intestinal segment was placed within the abdominal cavity,
carefully avoiding crimping or kinking of the segment. The
incision was then covered by saline-wetted paper towels. The
tested compounds (0.09 mM) were pamidronate (n ) 6),
alendronate (n ) 8), Pro-Phe (n ) 4), Pro-Phe-pamidronate (n
) 6) and Pro-Phe-alendronate (n ) 8). The permeability of Pro-
Phe-pamidronate was tested in the absence and in the pres-
ence of competitive inhibitors (n ) 6, 4 mM Pro-Phe). The
jejunal segments were perfused with a Sorensen’s phosphate
buffer solution (pH 7.4), containing one of the tested com-
pounds, with a flow rate of 0.19 mL/min using a peristaltic
pump (Ismatec, Switzerland). Samples were taken for 2 h in
10-min intervals after reaching steady state in the perfused
segment (after 30 min) and analyzed by scintillation counting.
Due to the double labeling of the compound, neither [14C]- nor
[3H]PEG-4000 could be examined to calculate water flux.
Therefore, water flux correction was accomplished by measur-
ing the inlet and outlet volume of the perfusate. Water
transport below 0.6%/cm of intestinal length was considered
to be normal and experiments with higher water transport
were not used in the determination of Peff. The concentration
ratios used for the permeability calculation were obtained
employing the following equation:
3
determinations of 14C (CO2) and H (H2O) counts in a mixture).
Counting of 14C and 3H enabled differentiation between Pro-
Phe-bisphosphonate and/or Phe-bisphosphonate and/or bis-
phosphonate on one hand, to Pro-Phe and/or Phe on the other.
Or a l Ad m in istr a tion . The tested compounds and dosages
were [14C]pamidronate (10 mg/kg, 1 µCi/mg), Pro-[3H]Phe-[14C]-
pamidronate (10 mg/kg of pamidronate, 14C 0.5 µCi/mg; 3H 2.6
µCi/mg), [14C]alendronate (10 mg/kg, 14C 1 µCi/mg) and Pro-
[3H]Phe-[14C]alendronate (10 mg/kg of alendronate, 14C 0.5 µCi/
mg; 3H 2.5 µCi/mg). Male Sabra rats weighing 250-300 g were
kept in metabolic cages for 1 week prior to the investigation
for acclimatization, with free access to water and food. Each
group of rats (n ) 6) received an oral dose of the tested
compound by a stomach tube after an overnight fast. 24 h later,
the animals were euthanized by an overexposure to ether, and
the tibia, femur, kidney, liver, intestine, muscle, urine and
feces were removed for radioactivity measurements. Radioac-
tivity levels in tissues were determined by combustion to
14CO2 and 3H2O by means of a sample oxidizer (Sample
Oxidizer, Packard, Meriden, CT) and liquid scintillation count-
ing, using appropriate standard curves (enabling separate
3
determinations of 14C (CO2) and H (H2O) counts in a mixture).
Counting of 14C and 3H enabled differentiation between Pro-

3650 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20
Cin′/Cout′ ) (Cin/Cout) × (Vin/Vout
Ezra et al.
Epithelial integrity of cell monolayers was routinely as-
sessed by the flux measurements of radiolabeled paracellular
marker [14C]mannitol (0.3 µCi/mL). [14C]Mannitol permeability
was not more than 4.1 × 10-7 cm/s from the apical to
basolateral direction and not more than 6.1 × 10-7 cm/s from
the basolateral to apical direction in each experiment. More-
over the TEER values were routinely measured using the
epithelial voltammeter (EVOM-G, World Precision Instru-
ments, Sarasota, FL). TEER values were 800 ( 90 Ω‚cm2 the
day before permeability studies. The TEER of the cell layers
was calculated using the following equation:
)
with Cin′/Cout′ as the volume-corrected concentration ratio, Cout
and Cin as the inlet and outlet perfusate concentrations,
respectively, and Vout and Vin as the inlet and outlet volumes
of the perfusate, respectively. For all experiments the inlet
and outlet volumes were measured with a micropipet (0.5-10
µL) up to a volume of 10 µL. This methodology had previously
been evaluated as a feasible substitution for nonabsorptive
markers.
The accuracy of the method was verified in preliminary
perfusion experiments by comparing water flux calculations
based on labeled PEG permeability (without the double-labeled
drug) to volume change of the perfusate. Water flux calculation
TEER ) (Rtotal - Rblanc)πr2
based on [14C]PEG-4000 recovery (PEGrec ) PEGout/PEGin
)
where Rtotal is the resistance measured, Rblanc is the resistance
of control filters without cells, and r is the radius of filter.
Radioactivity of the samples was determined using a liquid
scintillation counter (WinSpectral 1414 LSC, Wallac Co.,
Turku, Finland).
98 ( 5.3%) compared to that obtained by using volume change
of perfusate was in very good correlation. Moreover, we verified
this method of volume correction by determining the effective
permeability of various compounds (with different absorption
mechanisms and physicochemical properties). The results
obtained (L-arginine Peff ) 1.48 × 10-4 ( 1.581 × 10-5, n ) 6;
L-arginine + ouabaine Peff ) 6.73 × 10-5 ( 4.91 × 10-6, n ) 5;
PEG-4000 Peff ) 5.07 × 10-7 ( 4.13 × 10-7, n ) 5; and
colchicine Peff ) 4.82 × 10-5 ( 6.94 × 10-6, n ) 6) are in
accordance with the literature.35,36 The effective jejunal perme-
ability coefficient (Peff) was calculated according to the follow-
ing equation:36
Da ta An a lysis. The apparent permeability coefficients
(Papp) were calculated using the following equation:
Papp ) dQ/dt‚1/(AC0)
where dQ/dt is the permeability rate, A is the surface area of
the monolayer, and C0 is the initial donor solution concentra-
tion. For peptidyl prodrugs both the bisphosphonate and
proline-phenylalanine parts of the prodrug were quantified in
the receiver chamber and separate apparent permeability
coefficients were calculated.
Peff ) [Qin ln(Cin/Cout)]/2πrL
Sta tistica l An a lysis. The results are expressed as mean
( SD and were statistically evaluated by Mann-Whitney
U-test (p < 0.05).
where Qin is the perfusion flow, Cin/Cout (dpm/mL) is the volume
corrected concentration ratio, and 2πrL is the transfer surface
area, where L is the segment length and r is the radius (0.2
cm). Radioactivity levels in the specimens were determined
by liquid scintillation counting, using appropriate standard
curves as above.
Tr a n sp or t in th e Ca co-2 Cell Lin e. The tested com-
pounds were [14C]mannitol (51.5 mCi/mmol, purchased from
New England Nuclear Products, Boston, MA), [14C]alendronate
(1.0 µCi/mg), [14C]pamidronate (1.0 µCi/mg), Pro-[3H]Phe-[14C]-
alendronate ([3H] 2.3 µCi/mg, [14C] 0.5 µCi/mg), Pro-[3H]Phe-
[14C]pamidronate ([3H] 2.6 µCi/mg, [14C] 0.5 µCi/mg) and Pro-
[3H]Phe (6.35 µCi/mg).
Cell Cu ltu r e. Caco-2 cells (obtained from University of
California, San Francisco, CA) originating from a human
colorectal carcinoma were grown in 75 cm2 flasks at 7% CO2.
The culture medium consisted of DMEM supplemented with
10% heat-inactivated fetal bovine serum, 1% NEAA (nones-
sential amino acids), 2 mM L-glutamine, 100 IU/mL penicillin
and 100 µg/mL streptomycin. For the transport studies cells
were seeded at a density of 80 000 cells/ cm2 onto the rat tail
collagen (type I) coated Transwell polycarbonate filters (24-
mm diameter, 3.0-µm pore size) (Costar, Cambridge, MA). The
culture medium was changed every other day. Transport
studies were performed 21-25 days after seeding. Caco-2 cells
at passage number of 46-48 were used for experiments.
Tr a n sp or t Stu d ies. Transport studies were carried out in
Hank’s balanced salt solution (HBSS) (pH 7.4) containing 25
mM N-[2-hydroxyethyl]piperazine-N’-[2-ethanesulfonic acid]
(Hepes) as previously described.2 The cells grown on the
collagen-coated polycarbonate membranes were rinsed twice
with HBSS-Hepes solution before each experiment. At the
beginning of the experiment HBSS-Hepes solution was added
to the receiver chamber (either apical or basolateral side) and
the test solution to the donor chamber (either apical or
basolateral side). Bisphosphonates, their dipeptidyl prodrugs
and proline-phenylalanine were dissolved in HBSS-Hepes
solution to a concentration of 500 µM. The apical and baso-
lateral reservoir volumes were maintained at 1.5 and 2.6 mL,
respectively. The monolayers were incubated at 37 °C with
stirring. The samples (200 µL) were taken from the receiver
chamber at regular intervals up to 4 h and replaced with equal
volume of HBSS-Hepes solution.
Biocon ver sion . Bioconversion of Pro-[3H]Phe-[14C]pamidr-
onate was studied in intestinal mucosal homogenate and
artificial gastric juice. The cytosolic fraction of rat intestinal
mucosal was prepared according to the method of Bai et al.37
with some modification. The small intestine was detached and
its length was measured. An ice-cold solution of 0.15 M KCl
was pushed gently through the segment to clean out luminal
contents. The mucosa was scraped off and weighed, and 5 mL
of pH 7.8, 0.15 M Tris buffer was added to 1 g of mucosal
tissues. The tissues were homogenized in an ice bath at a speed
of 1140 rpm. The homogenate was then centrifuged at 4 °C,
108000g for 1 h. The supernatant fraction was harvested as
the enzyme source. The reaction solution consisted of 0.5 mL
of the supernatant and 0.5 mL of substrate solution (0.25, 0.5
and 1mM prodrug concentration), and the extent of overnight
hydrolysis (16 h) at 37 °C was determined. An appropriate
amount of 10% (w/v) trichloroacetic acid was used to stop the
hydrolysis. A solution of diluted phosphoric acid (pH 2) was
added 10 min later to achieve appropriate dilution prior to
centrifugation.
The extent of prodrug hydrolysis was determined by selec-
tive precipitation of the bisphosphonate as calcium salt. The
samples of the intestinal homogenate were combined with 100
µL of CaCl2 solution (CaCl2‚H2O, 150 mM) to precipitate the
bisphosphonate species (pamidronate, Phe-pamidronate or
Pro-Phe-pamidronate). In separate experiments we verified
that the amino acid Phe and the dipeptide, Pro-Phe, do not
precipitate in such conditions, and that the bisphosphonate is
completely precipitated. The specimens were centrifuged and
the dipeptide/amino acid (Pro-Phe, and/or Phe) amount in the
supernatant fraction was determined by radioactivity mea-
surements of 3H derivatives, which represented the fragments
cleaved off from pamidronate. The bioconversion of the prodrug
was determined from the ratio of 3H counts between the
precipitate and the supernatant.
Bioconversion of the prodrug was also studied in simulated
gastric juice. The artificial gastric juice solution (GJ S) con-
tained (in 1000 mL): 2 g NaCl, 80 mL 1 N HCl, and 3.2 g
pepsin at pH of 1.2 ( 0.1. Pro-Phe-pamidronate solution was
prepared in distilled water and was added in a 1:1 ratio to
the GJ S. The prodrug final concentrations were 0.5 and 0.25

Improving Bisphosphonate Oral Absorption
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20 3651
(2) Fleisch, H. A. Bisphosphonates: preclinical aspects and use in
osteoporosis. Ann. Med. 1997, 29, 55-62.
mM. After an overnight incubation at 37 °C, the extent of
hydrolysis was determined by selective precipitation of the
calcium-bisphosphonate complex as described previously.
In h ibition of Hyd r oxya p a tite P r ecip ita tion . The inhi-
bition of hydroxyapatite (HAP) precipitation formation in the
presence of Pro-Phe-pamidronate (0.1 and 1 mM) and Pro-Phe-
alendronate (0.1 and 1 mM) was studied as described previ-
ously.21 In brief, the HAP concentration product of calcium
(CaCl2‚2H2O) and phosphate (K2HPO4) in the incubated solu-
tions was 9 mM2, calcium 3.87 mM and phosphate 2.32 mM,
yielding a molar ratio of Ca/PO4 ) 1.67, as in HAP. Each salt
solution was prepared in 0.05 M Tris buffer, pH 7.4, and the
tested prodrug or drug was dissolved in the phosphate solution.
After 24 h of incubation, calcium concentration in the filtrate
was determined by atomic absorption spectroscopy.38
In h ibition of HAP Dissolu tion . HAP tablets (300 mg
HAP and 0.5% magnesium stearate) were prepared in a carver
press (10-mm diameter). The tablets were coated with beewax,
leaving only one surface exposed. The tablets were stirred at
37 °C overnight in a solution of Pro-Phe-pamidronate or
pamidronate (240 µM and 48 µM in 10 mL Tris buffer, pH
7.4. The HAP tablets with the adsorbed drug were transferred
to 10 mL acetate buffer pH 5, and stirred at 37 °C. At each
time point, aliquots of 1 mL were analyzed for dissolved
calcium by atomic absorption spectroscopy.38 To maintain
constant HAP:solution ratio, the dissolution medium was
replenished with 1 mL of acetate buffer after each sampling.
In h ibition of Ectop ic Ca lcifica tion . The anticalcification
effect of the prodrugs in-vivo was studied by examining the
inhibition of bioprosthetic tissue calcification implanted sub-
dermally in rats.21,30,39 Bioprosthetic heart valve tissue cusps
were prepared from bovine pericardium treated with glutaral-
dehyde, as described previously.38 Mini osmotic pumps (Alzet
2001, Alza, Stanford, CA) containing 0.014 M drug solution
delivering test compounds at 1.68 µmol/kg/day for 14 days,
were placed next to subcutaneous bioprosthetic tissue cusps
(1 × 1 cm) implanted in the dorsal part of each ether-
anesthetized rat. One tissue cusp, implanted subcutaneously
in the abdominal wall of each animal, served as a paired
control. An additional group of rats receiving bioprosthetic
tissue implants without treatment, served as an unpaired
control. Euthanasia was carried out by ether 14 days after
implantation, and the amount of calcium in the retrieved
tissues was determined by atomic absorption spectroscopy on
aliquots of HCl hydrolysates. The amount of calcium was
expressed as µg calcium/mg dry tissue weight. The control
values represent the average of the data of both control groups
since there was no difference between these groups.
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Sta tistics. The various parameters measured were ex-
pressed as mean ( SE and were statistically evaluated
according to the Mann-Whitney test (one-tailed, p < 0.05) or
Kruskal-Wallis and Dunn’s tests (two-tailed, p < 0.05).
Ack n ow led gm en t. This research was supported in
part by The Israel Science Foundation founded by the
Israel Academy of Sciences & Humanities (E.B.), by
grants (I-316-186/93 and I-383-188/94) from the Ger-
man-Israeli Foundation for Scientific Research and
Development (GIF, E.B., and G.G.), by TSRL Inc. (E.B.,
G.G., and G.L.A.), and by the Oppenheimer Fund of the
HUJ I. E. Breuer, A. Hoffman, and G. Golomb are
members of the David R. Bloom Center for Pharmacy
at The Hebrew University of J erusalem. The Caco-2
study was financially supported by the Technical De-
velopment Center of Finland (TEKES) and was a part
of the Biomacromolecule Research Program at the
University of Kuopio, Finland.
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