In vivo activities of farnesyl pyrophosphate synthase inhibitors against Leishmania donovani and Toxoplasma gondii.

Abstract

The in vivo activities of three bisphosphonates were determined against Leishmania donovani and Toxoplasma gondii. Alendronate was essentially inactive against both parasites. Pamidronate was active against L. donovani by intravenous administration. Risedronate had a 50% effective dosage of five 2.6-mg/kg of body weight intraperitoneal doses against L. donovani-infected mice but was less effective against T. gondii-infected mice.

Full text

Page 1

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 2002, p. 929–931
0066-4804/02/$04.00?0DOI: 10.1128/AAC.46.3.929–931.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 46, No. 3
In Vivo Activities of Farnesyl Pyrophosphate Synthase Inhibitors
against Leishmania donovani and Toxoplasma gondii
Vanessa Yardley,1Anis A. Khan,2† Michael B. Martin,3Teri R. Slifer,2Fausto G. Araujo,2
Silvia N. J. Moreno,4Roberto Docampo,4Simon L. Croft,1and Eric Oldfield3,5*
Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT,
United Kingdom1; Department of Infectious Diseases, Palo Alto Medical Foundation, Palo Alto, California 943012;
Department of Chemistry3and Department of Biophysics,5University of Illinois at Urbana-Champaign,
Urbana, Illinois 61801; and Laboratory of Molecular Parasitology, Department of Pathobiology,
University of Illinois at Urbana-Champaign, Urbana, Illinois 618024
Received 27 August 2001/Returned for modification 1 October 2001/Accepted 6 December 2001
The in vivo activities of three bisphosphonates were determined against Leishmania donovani and Toxoplasma
gondii. Alendronate was essentially inactive against both parasites. Pamidronate was active against L. donovani
by intravenous administration. Risedronate had a 50% effective dosage of five 2.6-mg/kg of body weight
intraperitoneal doses against L. donovani-infected mice but was less effective against T. gondii-infected mice.
Macrophages are the principal hosts or targets of the intra-
cellular forms of the parasitic protozoa Leishmania donovani
and Toxoplasma gondii, the causative agents of visceral leish-
maniasis and toxoplasmosis, respectively. Macrophage-like
cells, or osteoclasts, are also the targets of the bisphospho-
nate class of bone antiresorptive drugs, and in recent work
wefoundthatthebisphosphonatesalendronate(ALE)(Fosa-
max; Merck), pamidronate (PAM) (Aredia; Novartis), and
risedronate (RIS) (Actonel; Procter and Gamble) had consid-
erable activity against both L. donovani and T. gondii in vitro
(4). These protozoa are the cause of considerable mortality
and morbidity worldwide, so the development of novel chemo-
therapeutic approaches to their control is of importance, and
the use of bisphosphonates is of interest because they have
already been developed for other clinical indications. The first
in vivo results of the use of bisphosphonates against these two
organisms are described here.
For L. donovani in vivo assays, the parasite strain used was
MHOM/ET/67/L82. Female BALB/c mice (Tuck, United
Kingdom), 20 g, were infected with 2 ? 107amastigotes via
a lateral tail vein. Amastigotes were harvested from the spleen
of a golden hamster (Charles Rivers Ltd., Margate, United
Kingdom). Mice were randomly sorted into groups of five, and
after 7 days the levels of infection were checked, all groups
were weighed, and dosing commenced. In experiment 1,
groups of mice received either RIS at 20 mg/kg of body weight/
dose intraperitoneally (i.p.) or orally (p.o.), ALE at 20 mg/kg/
dose i.p. or p.o., or PAM at 20 mg/kg/dose i.p. or p.o. or over
a dose range of 10, 2, and 0.4 mg/kg/dose intravenously (i.v.).
All doses were given consecutively on days 7 to 11 of infection.
Another group of mice received sodium stibogluconate (Pen-
tostam) at 15 or 45 mg of SbV/kg/dose subcutaneously (s.c.) for
5 days as a positive control. In experiment 2, infected mice
received RIS at 15, 5, and 1.6 mg/kg/dose i.p. Positive and
negative control groups were also included. In experiment 3,
infected mice received RIS at 10, 3, and 1 mg/kg/dose i.p. or
Pro-Phe-ALE or ALE at a dosage of five 10-mg/kg doses p.o.
(10 mg/kg/dose p.o. ? 5) to determine any oral activity of the
former, a prodrug (3). Pro-Phe-ALE was the kind gift of Ger-
shon Golomb, Hebrew University, Jerusalem, Israel. At 14
days postinfection, all groups were sacrificed and liver impres-
sion smears were prepared and counted as described elsewhere
(2). For T. gondii in vivo assays, tissue cysts of strain C56 were
obtained as previously described (1). Swiss Webster female
mice (weight, 20 g at the beginning of each experiment) were
infected orally with 10 cysts (1). The bisphosphonates were
dissolved in phosphate-buffered saline (pH 6.8). Treatment
was initiated at 3 days postinfection and drugs were adminis-
tered once a day via i.p. injection for 10 days.
In the first set of experiments on the activities of the bisphos-
phonates in L. donovani, alendronate (ALE) proved inactive
when administered by either the p.o. or the i.p. route, with
toxic effects being observed following i.p. administration (Ta-
ble 1, footnote a). With PAM, our results, together with i.v.
results obtained with Trypanosoma cruzi-infected BALB/c mice
(6), suggested that i.v. administration was more effective, since
a 5-day treatment of 10-mg/kg doses i.v. gave a 91.8% suppres-
sion of liver amastigotes. When mice received doses of 2 mg/kg
i.v. ? 5 or less, the effect was greatly diminished. Using these
doses, a 50% effective dose of ?3.9 mg/kg was determined
compared with a 50% effective dose of 15.3 mg/kg s.c. ? 5 for
the standard drug, sodium stibogluconate (Table 1). A first
study with RIS indicated toxicity at 20 mg/kg, with two out of
five mice dying after administrations of 20 mg/kg ? 2 i.p. The
remaining three mice received one further dose, and there was
a 93.4% suppression of liver amastigotes in these animals (Ta-
ble 1). Oral administration was ineffective at 20 mg/kg ? 5,
consistent with the typically low (?0.5%) oral availability of
bisphosphonates.
In a second series of experiments, i.p. administration of RIS
* Corresponding author. Mailing address: Department of Chemis-
try, University of Illinois at Urbana-Champaign, 600 South Mathews
Ave., Urbana, IL 61801. Phone: (217) 333-3374. Fax: (217) 244-0997.
E-mail: eo@chad.scs.uiuc.edu.
† Present address: Global Metabolism and Investigative Sciences,
Pharmacia Corporation, 4901 Searle Pkwy., Skokie, IL 60077.
929

Page 2

was again studied and an 86.6% decrease in parasite burden at
5 mg/kg i.p. ? 5 was observed (Table 1). As toxicity (excessive
panting, horripilation) was also apparent at higher doses, a
split dose of 15 mg/kg ? 3 followed by 7.5 mg/kg ? 2 was then
used, resulting in a 99.1% decrease in parasite load.
In a third study, oral delivery of ALE was compared with
that of the ALE prodrug Pro-Phe-ALE (3), with small de-
creases in parasite burden being observed in both cases. A final
study of RIS i.p. administered over a dose range extended the
previous results and confirmed RIS as a lead compound for
visceral leishmaniasis, since there was a 73.6% decrease in
parasite burden at the lowest dosage of five 1-mg/kg i.p. doses,
97.6% at five 3-mg/kg i.p. doses, and a 99.6% decrease at 10
mg/kg ? 2 plus 5 mg/kg ? 3. In this study, some toxicity
(horripilation, panting) was initially seen at the higher dose but
no adverse reactions were seen at 3 mg/kg i.p. ? 5. These
results are of interest since they show that the nitrogen-
containing bisphosphonate RIS, a known farnesylpyrophos-
phate synthase inhibitor (5), can cure infections caused by the
parasitic protozoan L. donovani.
These results are consistent with in vitro parasite growth
inhibition studies of L. donovani reported previously (4). Spe-
cifically, RIS has a 2.3 ?M 50% inhibitory concentration (IC50)
against L. donovani amastigotes, while ALE and PAM are
much less effective (82.5 and ?300 ?M IC50s, respectively) (4).
The toxicity effects of ALE, PAM, and RIS are also consistent
with in vitro therapeutic index (T.I.) estimates, where the T.I.
is defined as 50% lethal dose (LD50) (human nasopharynx
carcinoma)/IC50(L. donovani). LD50refers to the concentra-
tion of drug (?M) causing a 50% reduction in viability in the
human cell line (M. B. Martin, H. Kendrick, K. de Luca-
Fradley, J. C. Lewis, J. S. Grimley, E. M. van Brussel, J. R.
Olsen, S. L. Croft, and E. Oldfield, unpublished data), and IC50
represents the decrease in L. donovani growth in vitro (from
reference 4). Specifically, ALE and PAM are the least effective
drugs in the BALB/c mouse experiments (T.I. ? 1.76 and ?
0.52, respectively), while RIS is the most effective (T.I. ? 108).
For Toxoplasma gondii, an initial series of experiments were
conducted using ALE, PAM, or RIS doses ranging from 1 to 20
mg/kg i.p. ? 10. All untreated control mice orally infected with
tissue cysts died by day 14, with a mean survival time of 11.2
days. The administration of PAM and ALE offered no protec-
tion against death: all infected animals treated with either
PAM or ALE died before the controls. However, RIS at 10
mg/kg i.p. ? 10 provided a 40% survival rate (P ? 0.01), with
a mean survival time of 19.6 days. We therefore carried out
three additional trials with RIS in order to verify the in vivo
activity seen in the first trial. Figure 1 shows the cumulative
results of all four in vivo RIS trials (n ? a total of 40 treated
mice) against T. gondii. All untreated control mice orally in-
fected with tissue cysts died by day 16, with a mean survival
time of 12.7 days (n ? 20). RIS at 10 mg/kg i.p. ? 10 (n ? 40)
provided a 35% survival rate (P ? 6 ? 10?7), with a mean
survival time of 20.3 days, while RIS at 20 mg/kg i.p. ? 10
(n ? 40) provided a 55% survival rate (P ? 4 ? 10?4) with a
mean survival time of 22.1 days.
In these studies, the significant in vitro activities of PAM and
ALE reported previously (4) did not translate into curative in
vivo activities. The in vitro IC50values for PAM and ALE are
35.6 ?M and 25 ?M, respectively (4). The LD50s of these
compounds are both ?150 ?M in the mammalian cell model
used, which translates into rather low therapeutic indices
TABLE 1. In vivo activity of nitrogen-containing bisphosphonates
and a bisphosphonate prodrug against Leishmania donovani
DrugDosing regimen Inhibition (%)
Expt 1
ALE 20 mg/kg p.o. ? 5
20 mg/kg i.p. ? 4
0
14.7a
PAM20 mg/kg p.o. ? 3, 10 mg/kg i.p. ? 2
20 mg/kg i.p. ? 5
10 mg/kg i.v. ? 5
2 mg/kg i.v. ? 5
0.4 mg/kg i.v. ? 5
13.5b
0
91.8
15.0
0
RIS20 mg/kg p.o. ? 5
20 mg/kg i.p. ? 3
0
93.4c
Pentostam45 mg SbV/kg s.c. ? 5
15 mg SbV/kg s.c. ? 5
94.7
48.8
Expt 2
RIS 15 mg/kg i.p. ? 3, 7.5 mg/kg i.p. ? 2
5 mg/kg i.p. ? 5
1.6 mg/kg i.p. ? 5
99.1d
86.6
19.5
Pentostam15 mg SbV/kg s.c. ? 5
51.73
Expt 3
Pro-Phe-ALE 10 mg/kg p.o. ? 5 9.2
ALE10 mg/kg p.o. ? 5 21.1
RIS 10 mg/kg i.p. ? 2, 5 mg/kg i.p. ? 3
3 mg/kg i.p. ? 5
1 mg/kg i.p. ? 5
99.6e
97.6
73.6
Pentostam15 mg SbV/kg s.c. ? 5
17.1
aOne of five mice died after the final dose. The percentage of inhibition refers
to the surviving mice.
bMice received 1/2 dose on 4th and 5th days of dosing due to apparent toxicity
(excessive panting and horripilation).
cTwo of five mice died after two doses. The remaining three mice received
three doses in total. The percentage of inhibition refers to the surviving mice.
dThis group received dosages of 15 mg/kg of body weight ? 3 and then 7.5
mg/kg ? 2, due to apparent toxicity.
eInitially toxic to mice at 10 mg/kg; therefore, dose was halved for last 3 days.
FIG. 1. Cumulative survival results for T. gondii-infected mice after
RIS administration in vivo for two different RIS concentrations. I,
diluent (n ? 20); {, 10 mg/kg (n ? 40); ?, 20 mg/kg (n ? 40). Kaplan-
Meier statistics were used to deduce standard error bars.
930 NOTESANTIMICROB. AGENTS CHEMOTHER.

Page 3

(5.8 for ALE and 4.4 for PAM), and in this murine model of
toxoplasmosis, i.p. administration of either drug provided
no protection against infection. However, in the case of RIS
(IC50? 0.49 ?M), there was a significant increase in mouse
survival following T. gondii infection if RIS was administered.
This is consistent with the high therapeutic index (510) seen
with RIS in the in vitro experiments.
These results indicate that the potent nitrogen-containing
bisphosphonate drug RIS is a promising lead compound for
development as a novel antiparasitic agent. RIS has a relatively
broad spectrum of antiparasitic activity in in vitro testing (4)
and has also been found to be the most potent inhibitor of
L. donovani and T. gondii growth in vitro, in addition to having
a relatively low overall cytotoxicity. The drug has a high ther-
apeutic index and can give ?99% decreases in parasite burden
in L. donovani-infected mice. In cutaneous leishmaniases, oth-
er bisphosphonates may also be effective; for example, in a
Leishmania mexicana amazonensis BALB/c model of infec-
tion, PAM (10 mg/kg i.p. ? 5) effects a parasitological cure (N.
Rodriguez, B. N. Bailey, M. B. Martin, E. Oldfield, J. A.
Urbina, and R. Docampo, unpublished data), confirming that
the nitrogen-containing bisphosphonates are a class of com-
pounds worthy of further investigation as antiparasitic agents.
This work was supported in part by the United States Public Health
Service (National Institutes of Health Grant GM-50694 to E.O. and
AI04717 and N01-AI-35174 to F.G.A.), by the UNDP/World Bank/
WHO Special Programme for Research and Training in Tropical Dis-
eases (TDR) (E.O., V.Y., S.L.C., R.D.), and by the American Heart
Association, Midwest Affiliate (E.O.) M.B.M. is an American Heart
Association Predoctoral Fellow, Midwest Affiliate. S.N.J.M. is a Bur-
roughs Welcome New Investigator in Molecular Parasitology.
We also thank Gershon Golomb for providing the Pro-Phe-ALE.
REFERENCES
1. Araujo, F. G., A. A. Khan, T. L. Slifer, A. Bryskier, and J. S. Remington. 1997.
The ketolide antibiotics HMR 3647 and HMR 3004 are active against Toxo-
plasma gondii in vitro and in murine models of infection. Antimicrob. Agents
Chemother. 41:2137–2140.
2. Croft, S. L., D. Snowdon, and V. Yardley. 1996. The activities of four anti-
cancer alkyllysophospholipids against Leishmania donovani, Trypanosoma
cruzi and Trypanosoma brucei. J. Antimicrob. Chemother. 38:1041–1047.
3. Ezra, A., A. Hoffman, E. Breuer, I. S. Alferiev, J. Monkkonen, N. El Hanany-
Rozen, G. Weiss, D. Stepenksy, I. Gati, H. Cohen, S. Tormalehto, G. L.
Amidon, and G. Golomb. 2000. A peptide prodrug approach for improving
bisphosphonate oral absorption. J. Med. Chem. 43:3641–3652.
4. Martin, M. B., J. S. Grimley, J. C. Lewis, H. T. Heath III, B. N. Bailey, H.
Kendrick, V. Yardley, A. Caldera, R. Lira, J. A. Urbina, S. N. J. Moreno, R.
Docampo, S. L. Croft, and E. Oldfield. 2001. Bisphosphonates inhibit the
growthofTrypanosomabrucei,Trypanosomacruzi,Leishmaniadonovani,Toxo-
plasma gondii and Plasmodium falciparum: a potential route to chemotherapy.
J. Med. Chem. 44:909–916.
5. Montalvetti, A., B. N. Bailey, M. B. Martin, G. W. Severin, E. Oldfield, and R.
Docampo. 2001. Bisphosphonates are potent inhibitors of Trypanosoma cruzi
farnesyl pyrophosphate synthase. J. Biol. Chem. 276:33930–33937.
6. Urbina, J. A., B. Moreno, S. Vierkotter, E. Oldfield, G. Payares, C. Sanoja,
B. N. Bailey, W. Yan, D. A. Scott, S. N. Moreno, and R. Docampo. 1999.
Trypanosoma cruzi contains major pyrophosphate stores, and its growth in
vitro and in vivo is blocked by pyrophosphate analogs. J. Biol. Chem. 274:
33609–33615.
VOL. 46, 2002NOTES 931