U.S. patent application number 10/119502 was filed with the patent office on 2003-02-13 for antifungal and antiparasitic compounds.
Invention is credited to Ayafor, Johnson F., Bacchi, Cyrus, Iwu, Maurice M., Jackson, Joan E., Okunji, Christopher O., Tally, John D. JR..
Application Number | 20030032578 10/119502 |
Document ID | / |
Family ID | 26793519 |
Filed Date | 2003-02-13 |
United States Patent
Application |
20030032578 |
Kind Code |
A1 |
Jackson, Joan E. ; et
al. |
February 13, 2003 |
Antifungal and antiparasitic compounds
Abstract
Novel antiparasitic and antifungal compositions are disclosed.
The antiparasitic and antifungal compositions are useful for human
and veterinary therapy for the treatment and/or prevention of
parasitic infection. Also disclosed are novel mechanisms of
identifying antifungal and antiparasitic compositions by their
biochemical action on lipid synthesis and/or metabolism and/or
excretion.
Inventors: |
Jackson, Joan E.;
(Rockville, MD) ; Iwu, Maurice M.; (Silver Spring,
MD) ; Okunji, Christopher O.; (Silver Spring, MD)
; Bacchi, Cyrus; (East North Port, NY) ; Tally,
John D. JR.; (Washington, DC) ; Ayafor, Johnson
F.; (Dechang, CM) |
Correspondence
Address: |
Attn: MCMR-JA (Ms. Arwine)
Elizabeth Arwine, Esq.
US Army Medical Research & Material Command
504 Scott Street
Fort Detrick
MD
21701-5012
US
|
Family ID: |
26793519 |
Appl. No.: |
10/119502 |
Filed: |
April 10, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10119502 |
Apr 10, 2002 |
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09382128 |
Aug 24, 1999 |
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6403576 |
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60097672 |
Aug 24, 1998 |
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Current U.S.
Class: |
514/1 |
Current CPC
Class: |
A61K 31/55 20130101 |
Class at
Publication: |
514/1 |
International
Class: |
A61K 031/00; A61K
031/55; A61K 031/553; A61K 031/554 |
Claims
What is claimed is:
1. An antiparasitic composition comprising an inhibitor of
cholesterol synthesis.
2. An antiparasitic composition comprising an inhibitor of
cholesterol metabolism.
3. An antiparasitic composition comprising an inhibitor of
cholesterol excretion.
4. An antiparasitic composition comprising at least one inhibitor
selected from the group consisting of inhibitor of cholesterol
synthesis, inhibitor of cholesterol metabolism, and inhibitor of
cholesterol excretion.
5. A method for treating an individual including a human with a
parasitic infection comprising administering to said individual a
cholesterol synthesis inhibitor in a pharmaceutically effective
amount, in a pharmaceutically effective excipient.
6. A method for treating an individual including a human with a
parasitic infection comprising administering to said individual a
cholesterol metabolism inhibitor in a pharmaceutically effective
amount, in a pharmaceutically effective excipient.
7. A method for treating an individual including a human with a
parasitic infection comprising administering to said individual a
cholesterol excretion inhibitor in a pharmaceutically effective
amount, in a pharmaceutically effective excipient.
8. A method for preventing parasitic infection in an animal
including a human comprising administering to said animal a
cholesterol synthesis inhibitor in a pharmaceutically effective
amount, in a pharmaceutically effective excipient.
9. A method for preventing parasitic infection in an animal
including a human comprising administering to said animal a
cholesterol metabolism inhibitor in a pharmaceutically effective
amount, in a pharmaceutically effective excipient.
10. A method for preventing parasitic infection in an animal
including a human comprising administering to said animal a
cholesterol excretion inhibitor in a pharmaceutically effective
amount, in a pharmaceutically effective excipient.
11. A method according to claim 5 wherein said administration is
selected from the group consisting of oral, topical and
parenteral.
12. A method according to claim 6 wherein said administration is
selected from the group consisting of oral, topical and
parenteral.
13. A method according to claim 7 wherein said administration is
selected from the group consisting of oral, topical and
parenteral.
14. A method according to claim 8 wherein said administration is
selected from the group consisting of oral, topical and
parenteral.
15. A method according to claim 9 wherein said administration is
selected from the group consisting of oral, topical and
parenteral.
16. A method according to claim 10 wherein said administration is
selected from the group consisting of oral, topical and
parenteral.
17. An antiparasitic composition comprising an agent which
interferes with parasite cholesterol uptake.
18. An antiprasitic composition comprising an inhibitory agent
selected from the group consisting of inhibitors of cholesterol
production in host, inhibitors of cholesterol transport to monocyte
of host, inhibitors of cholesterol delivery to the parasite,
inhibitors of choline production, inhibitors of HMG-COA reductase,
inhibitors of squalene oxidase, inhibitors of squalene synthetase,
inhibitors of 14alpha-demethylase, inhibitors of bile acids
resorption, inhibitors of butyrate, anticance hormone
agonists/antagonists, hypocholesteremics, or a combination
thereof.
19. An antiparasitic composition comprising one or more
hypocholesteremic.
20. The antiparasitic composition of claim 19 wherein the
hypocholesteremic is beta-carotene or lycopene.
21. The antiparasitic composition of claim 19 wherein the
hypocholesteremic is an estrogen agonist or antagonist.
22. A method for treating an individual with a parasitic infection
comprising administering to said individual an antiparasitic
composition of claim 18 in a pharmaceutically effective amount, in
a pharmaceutically effective excipient.
23. A method for preventing parasitic infection in an individual
comprising administering to said individual an antiparasitic
composition of claim 18 in a pharmaceutically effective amount, in
a pharmaceutically effective excipient.
24. A method according to claim 22 wherein said administration is
selected from the group consisting of oral, topical, and
parenteral.
25. An antiparasitic composition comprising an antiparasitic
effective amount of at least one compound which inhibits a
parasite's cholesterol biosynthesis, metabolism, and/or
excretion.
26. An antiparasitic composition comprising an antiparasitic
effective amount of a combination of compounds selected from the
group consisting of inhibitors of cholesterol production in host,
inhibitors of cholesterol transport to monocyte of host, inhibitors
of cholesterol delivery to the parasite, inhibitors of choline
production, inhibitors of HMG-CoA reductase, inhibitors of squalene
oxidase, inhibitors of squalene synthetase, inhibitors of
14alpha-demethylase, inhibitors of bile acids resorption,
inhibitors of butyrate, anticance hormone agonists/antagonists,
hypocholesteremics,
Description
FIELD OF THE INVENTION
[0001] Compounds are described which represent novel, efficacious,
and less toxic alternatives to current antiparasitic/antifungal
treatments. Compounds having action via the biochemical mechanism
of inhibition of lipid synthesis and/or metabolism and/or
excretion, either by direct or indirect inhibition, will have
either singly or in combination antiparasite/antifungal activity.
Such compounds, in most cases, are not chemically related by
structure or chemical class to each other. The compounds are
identified as antiparasitics and/or antifungals based on mechanism
of physiologic action. Data supporting "novel use" as
antiparasite/antifungal compounds are given. Many compounds herein
described are FDA-approved and marketed for human use for
nonparasitic/nonfungal indications. Thus, the human
pharmacokinetics for oral absorption, elimination rates/mechanisms,
and dose-related toxicity are known.
INTRODUCTION
[0002] Status of Leishmaniasis, Trypanosomiasis, and
Trichomoniasis
[0003] Current drugs most frequently used to treat leishmaniasis
all require parenteral administration, date back 40->50 years,
and all have such severe side-effects that treatment only in a
hospital setting is recommended (Bryceson, 1968, East African Med J
45, 110-117; Bryceson, A., 1987, The Leishmaniases in Biology and
Medicine, Vol II Clinical Aspects and Control, Academic Press, New
York, pp. 847-907). No antileishmanial is Food and Drug
Administration (FDA) approved and there is no chemoprophylaxis for
any leishmanial disease. Topical treatment for leishmanial disease
is not effective even for cutaneous disease forms because
leishmaniasis is a systemic disease (Neva, et al., 1997, Trans R
Soc Trop Med Hyg 91, 473-475). There is no general vaccine for
leishmaniases, although a live vaccine is used in the Middle East
for certain Leishmania (Leishmania) tropica/Leishmania (Leishmania)
major to prevent facial scarring. Drug resistance is so severe in
certain endemic regions that thousands are dying in India of
untreatable, multidrug resistant visceral leishmaniasis; and in
Northern Africa as a result of malnutrition exacerbated disease
(Cerf, et al., 1987, J Inf Dis 156, 1030-1033; de Beer, et al.,
1991, Am J Trop Med Hyg 44, 283-289; Sundar, 1997, Acta Parasitol
Turicica 21, suppl 1, 128).
[0004] Immunodeficiency, either as the result of leishmanial
tubercular- or HIV coinfections, poses serious therapeutic
difficulties as leishmanial coinfection is reported to potentiate
the pathology of both these bacterial and viral infections (Alvar,
et al., 1997, Clin Microbiol Rev 10, 298-319; Bernier R, et al.,
1995, J Virol 69, 7282-7285; Bryceson, 1987, supra;
Faraut-Gamarelli, et. al., 1997, Antimicrob Agents Chemother 41,
827-830). Global travel and commerce result in patients having
complex disease exposure history, and transportation of leishmanial
parasites far from their anticipated endemic regions making both
diagnosis and patient management difficult (Albrecht, et al., 1996,
Arch Pathol Lab Med 120, 189-198). Leishmaniases have an annual
incidence of 2-3 million new cases per year with 12 million
infected and 350 million at risk in 88 countries worldwide (Croft,
1988, Trends Pharmacol Sci 9, 376-381; World Report on Tropical
Diseases, 1990). The need for a orally administered antileishmanial
of low toxicity is critical.
[0005] Two major groups of diseases caused by flagellate protozoa
are African sleeping sickness (Trypanosoma brucei spp.) and
trichomoniasis (Trichomonas/Tri trichomonas) exhibited as
trichomoniasis vaginalis and trichomoniasis foetus.
[0006] African trypanosomiasis affects both domestic and wild
animals as well as humans in mainly rural settings (Kuzoe, 1993,
Acta Tropica 54, 153-162; World Health Organization (WHO), 1995,
Tropical Disease Research, Twelfth Programme Report, Geneva
Switzerland) while trichomoniasis is a cosmopolitan disease in men
as well as women, and a threat to cattle breeding in most
agricultural areas of the world (Hammill, 1989, Obstet Gynecol Clin
North Am 16, 531-540; Levine, 1985, Veterinary Protozoology. Iowa
State Univ. Press, Ames, pp 59-79). Treatment of the organisms
causing these diseases presents problems, in part, due to the
toxicity of existing agents, and the development of resistance to
existing drugs (Kuzoe, 1993, supra; Lossick, 1989, Trichomonads
Parasite in Humans. Springer-Verlag, New York, pp 324-341).
[0007] African trypanosomiasis is endemic in over 10 million square
kilometers of sub-Saharan Africa, affecting humans and all
domesticated livestock (WHO, 1995, supra). There are an estimated
25,000 new cases of human disease yearly and an animal incidence of
250-300,000 cases but these estimates are low, based on recent
civil unrest and lapses in local tsetse fly control and medical
surveillance (WHO, 1995, supra). The primary drugs for human and
veterinary trypanosomiasis have been in use for >50 years.
Resistance is spreading, especially to the only available agent for
late stage central nervous system (CNS) human disease, melarsoprol
(van Nieuwenhove, 1992, Ann Soc Belg Med Trop 72, 39-51; Kuzoe,
1993, supra). Melarsoprol is also toxic, with a 3-5% incidence of
cerebral episodes reported (Pepin and Milord 1994, Adv Parasitol
33, 2-47; Wery, 1994, Int j Antimicrob Agents 4, 227-238).
Veterinary trypanocides include diminazene (Berenil.RTM.) and
isometamidium (Samorin.RTM.) which are used prophylactically for
control of disease in cattle herds (WHO, 1995, supra; Kaminsky et
al., 1993, Acta Tropica 54, 19-30). Resistance to both agents has
been documented in field studies (Kuzoe, 1993, supra; Schoenfeld et
al., 1987, Trop Med Parasitol 38, 117-180; Williamson, 1970, The
African Typanosomiases. Allen & Unwin, London, pp 125-224). For
these reasons, there is an urgent need to develop new
trypanocides.
[0008] Trichomonas vaginalis is one of the most prevalent sexually
transmitted pathogen of the human urogenital tract. It infects the
vaginal epithelium, causing severe irritation and the development
of a discharge. In addition to social distress caused by the
disease, recent evidence suggests a high incidence rate between
cervical cancer and trichomoniasis (Gram et al., 1992, Cancer
Causes and Control 3, 231-236). The disease is widespread, with
about 3 million cases in women annually in the United States alone
(Hammill, 1989, supra). Chemotherapy for human trichomoniasis
relies on a group of 5'-nitroimidazoles, with metronidazole
(Flagyl.RTM.) being the most utilized. In the United States,
metronidazole is the only available agent, although other
derivatives are used in Europe and other areas. Since metronidazole
has been in continuous use since 1955, there has been increasing
reports of metronidazole-resistant vaginitis (Meingassner &
Thurner, 1979, Antimicrob Agents Chemother 15, 254-258; Wong et
al., 1990, Australia-New Zealand J Obstet Gynecol 30, 169-171;
Voolman & Boreham, 1993, Med J Australia 159, 490). Because of
its potential to produce free radicals upon reduction, it is
potentially mutagenic and not given to pregnant women (Lossick,
1989, supra). At present, there is no alternative to the
5'-nitroimidazoles for therapy of metronidazole-refractory disease,
nor for treatment of pregnant women.
[0009] Trichomonas foetus is the agent of bovine trichomoniasis,
causing reproductive failure. Parasites are spread by infected
bulls, multiply in the vagina and invade the cervix and uterus. One
to 16 weeks after breeding, abortion of the fetus occurs (Levine,
1985, supra). If the placenta and fetal membranes are eliminated
following abortion, the cow may spontaneously recover. If some of
these tissues remain inside the animals, permanent sterility may
result. There is no satisfactory treatment for diseased cows, while
treatment of bulls is tedious and expensive. Aminoquinuride
(Surfen.RTM.) or acriflavine (Trypaflavine.RTM.) may be used
topically, with dimetridazole injected into the urethra. Unless the
bull is valuable, it is usually destroyed (Levine, 1985, supra).
The disease is common in open range breeding ranches and may reach
epidemic levels. In Australia, 40-65% of cattle were reported to be
infected, while the, prevalence in California was reported to be
14% (Yule et al., 1989, Parasitol Today 5. 373-377). The economic
losses due to bovine trichomoniasis have been estimated to be
$665/infected dairy cow, while the widespread prevalence of the
disease would account for tens of millions of dollars annually
(Yule et al., 1989, Parasitol Today 5, 373-377). The overall
situation for chemotherapy of trichomoniasis therefore, is the
reliance on a single drug as drug class for chemotherapy of human
disease, and no effective control measures for bovine
trichomoniasis.
SUMMARY OF THE INVENTION
[0010] Preliminary evidence from our ethnomedical and
ethnobotanical drug discovery research as well as background
literature describing different aspects of the parasite's sterol
pathway and cholesterol requirements and importance to parasite
survival, has led to the discovery of compounds chosen on the basis
of their physiological function on different parts of the sterol
synthesis, and/or excretion, and/or metabolism which offer
potential chemotherapeutic target(s) having low toxic potential for
man. Several of these compounds have been tested for their
antiparasitic/antifungal activity as described in the Examples.
[0011] The following is a brief summary of the background and data
which led to the discovery of the antiparasitic/antifungal
compounds of the present invention.
[0012] Lipids comprise up to 15% of the total dry weight of
Leishmania spp. (Meyer and Holz, 1966, J Biol Chem 241, 5000-5007;
Beach, et al., 1979, J Parasitol 65, 203-216; Fish, et al., 1981,
Mol Biochem Parasitol 3, 103-116). Lipid metabolism is critical to
parasite membrane transport, cell replication, and, therefore, to
survival. The lipid metabolism of Leishmania spp. including
precursors, synthetic pathways, regulator molecules, and end
products for membrane fatty acids, lipids, and sterols is known to
mimic parts of fungal, bacterial-, plant-, and human lipid
pathways, while completely duplicating none. Because leishmanial
lipid metabolism is unique among organisms, genetically conserved
(Wendt, et al., 1997, Science 277, 1811-1815), and
biochemically-tightly regulated (Thompson, 1992, The Regulation of
Membrane Lipid Metabolism. CRC Press, Ann Arbor, pp 230), the
sterol pathway has the potential to provide us chemotherapeutic
targets not duplicated in humans (drug development).
[0013] Leishmania share with plants (and animals) that they rely on
mevalonic acid as a precursor for de novo sterol synthesis (Holz,
1985, Leishmaniasis. Elsevier, N.Y., pp 79-92; Thimann, 1977,
Hormone Action in the Life of Plants. University of Massachusets
press, Amherst, pp. 448; Thompson, 1992, supra) However, the major
sterol of leishmanial and fungal membranes, synthesized de novo by
these parasites, is not cholesterol (like humans), but a
24-substituted sterol (ergosterol or episterol or provitamin D2).
Ergosterol is synthesized by these parasites de novo from
acetylCoA, to mevalonate, to squalene, to lanosterol, and 4 steps
later to ergosterol (Holz, 1985, supra). Coppens and Courtoy (1995,
Mol Biochem Parasitol 73, 179-188) showed that procyclics of T.
brucei normally contain ergosterol synthesized de novo, a pathway
shared with Leishmania.
[0014] However, Leishmania require cholesterol. Unlike man, but
like closely related Kinetoplastid parasites, of the genus
Trypanbsoma, Leishmania "salvage" cholesterol from their
environment, i.e., from macrophages and monocytes (the
LDL/cholesterol plasma clearance cells) in the mammalian
reticuloendothelial system. Free cholesterol and free fatty acids
do not occur normally in plasma. The cholesterol esters of fatty
acids, which are by themselves insoluble in plasma, are located in
the low density lipoprotein, LDL, as a nonpolar core surrounded
with a polar shell of phospholipids, apoprotein, and unesterified
cholesterol, thus ensuring solubilization and transport (Ormerod
& Venkatesan, 1982, Microbiol Rev 46, 296-307; Thompson, 1992,
supra). Leishmania reside in mononuclear macrophages, which
comprise the major part of low-density lipoprotein (LDL) plasma
clearance system via both receptor and receptor-independent
mechanisms (Goldstein & Brown, 1976, Curr Top Cell Regul 11,
147-181; 1977, Ann Rev Biochem 46, 897-930; Weisgraber, et al.,
1978, J Biol Chem 253, 9053-9062; Pangburn, et al., 1981, J Biol
Chem 256, 3340-3347; Bilheimer, et al, 1982, Proc Natl Acad Sci USA
79, 3305-3309; Haughan, et al., 1992, Biochem Pharmacol 44,
2199-2206). Transport of LDL-cholesterol via either or both
mechanisms into infected monocytes would thus allow leishmanial
parasites to meet their cholesterol requirement. Drugs which
interrupt the quantity, transport, or delivery of cholesterol to
the parasite would have potential to adversely affect leishmanial
survival.
[0015] There are marked metabolic similarities between leishmanial
and trypanosomal lipid acquisition and metabolism. Bloodstream
forms of Trypanosoma brucei spp. can ingest particulate fat (Wooten
& Halsey, 1957, Parasitol 47, 427-431), and, like Leishmania,
Trypanosoma brucei rhodesiense depends on the cholesterol of their
habitat (Dixon et al., 1972, Comp Biochem Physiol 41B, 1-18).
[0016] Coppens and colleagues (1995, Mol Biochem Parasitol 73,
179-188) showed that the enzyme inhibitor, synvinolin (simvastatin
or Zocor.RTM.), potentiates growth inhibition of Trypanosoma brucei
in the presence of drugs interfering with the exogenous supply of
cholesterol; and conversely, growth inhibition by synvinolin can be
reversed by LDL, mevalonate, squalene or cholesterol. Coppens and
Courtoy (1995, supra) showed that procyclics of T. brucei spp.
normally incorporate exogenous cholesterol in their membranes.
These investigators further demonstrated that growth of the
culture-adapted trypanosomes is accelerated by supplementation of
the medium with low density lipoprotein (LDL) particles which were
endocytosed by the parasites via a receptor-mediated mechanism.
[0017] We observed that traditional medical herbal therapies,
containing plant sterols having the cholestane backbone but with
hydrophillic substitutent side chains, first destabilized then
killed parasites in vitro in a dose-dependent manner. Chemical
analyses of the structure of the antiparasitic active moieties from
these plants (>70 tested) most frequently revealed an
isoprenoid, terpenoid, or steroidal structure resembling but not
duplicating normal mammalian sterolgenic precursors. It is known,
as previously discussed, that Leishmania spp. and African
Trypanosoma spp. take up cholesterol and any cholestane-backbone
molecule (Dixon, et al., 1972, supra; Haughan, et al. 1995, supra).
We believe that substitute "plant cholesterol-like" molecules serve
to destabilize parasites' membranes because of either addition of
new hydrophillic sidegroups; or replacement of typically
hydrophobic side-groups with more hydrophillic side-groups. These
observations, in addition to the knowledge of the importance of
cholesterol and cholesterol synthesis in the organism, appeared to
validate the use of these medicinal plants as herbal remedies for
treatment of protozoan parasitic infections.
[0018] Therefore, at several points within the sterol synthesis and
cholesterol salvage pathways, we have identified molecules
chemically or functionally similar to the natural component, but
which act to shut-down leishmanial function.
[0019] Therefore, it is one object of the present invention to
provide a novel method for identifying compounds having
antiparasitic and antifungal activity based on the physiological
action of the compounds in the sterol synthesis and/or metabolism,
and/or excretion pathway of the parasite.
[0020] It is also an object of the present invention to provide a
novel method for identifying antifungal and antiparasitic compounds
by their ability to inhibit cholesterol synthesis and/or metabolism
and/or excretion, directly or indirectly.
[0021] It is further an object of the present invention to provide
novel antiparasitic and antifungal agents which are capable of oral
administration, and are efficacious and less toxic alternatives to
agents heretofore used for the treatment of fungal and/or parasitic
infection in humans and animals.
[0022] A still further object of the present invention is to
provide a novel method of using existing compounds not previously
known to have antifungal or antiparasitic activity for the
prevention and/or treatment of fungal or parasitic infection in
humans and animals.
[0023] It is also an object of the present invention to provide
antiparasitic and antifungal compositions for either prophylactic
or field treatment.
[0024] A further object of the present invention includes the
combined therapy that can be obtained by treating patients with
leishmania, trichomoniasis, or trypanosomiasis, with a combination
of the compounds of the present invention, preferbly the
combination is chosen such that compounds which inhibit different
parts of the cholesterol pathway are combined.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] These and other features, aspects, and advantages of the
present invention will become better understood with reference to
the following description and appended claims, and accompanying
drawings where:
[0026] FIG. 1. A schematic representation of the mechanism of
cholesterol regulation indicating eleven types of inhibitors of
lipid metabolism, synthesis, or excretion having
antiparasitic/antifungal properties (bold cap letters).
DETAILED DESCRIPTION
[0027] Forty-two medicinal plants were identified as having
antileishmanial properties from ethnomedical studies and either
antileishmanial/antifungal properties from ethnobotanical research.
Fifty percent (21/42) plants and 59/121 extracts tested showed in
vitro antileishmanial activity. The chemical isolation strategy
focused preferentially on isolation of di- and tri-terpenes
(sterol-like) compounds which seemed to contain highly active
(>90% cidal in vitro) antileishmanial compounds. The first
compound to be characterized was a spirostanol saponin,
Mannispirotan A, isolated from the fruit pulp of Dracaena manii
(Okunji et al., 1990, Int J Crude Drug Res 28, 193-199) Study of
the structure (shown below at `A`) revealed a resemblance to a
sterol nuclei structure. 1
Spirostanol Saponin
[0028] Four additional highly active extracts have been purified
and their structures, which include more than 25 separate
compounds, determined. Most are compounds that have chemical
congeners, isoprenoids, di- and triterpenoids common to lipid
metabolism; a few are berberine-like or -dimers presented in U.S.
Pat. No. 5,290,553, to Iwu, et al., 1994. All documents cited
herein supra or infra are incorporated in their entirety by
reference thereto. Knowledge of structure activity relationship
(SAR) has allowed us to formulate hypotheses for the mechanism of
antiparasite physiologic inhibition.
[0029] When additional plant extracts were examined, and additional
active structures elucidated, namely, Sakuretin from Eupatorium
odoratum, Labdane-dial from Aframomum danielli, and Afromomum
aulocacaxpus, unexpectedly, the structures of these compounds did
not resemble cholesterol, but instead resembled Vitamin D2 and
possible parts of a squalene isoprenoid structure as it is
cyclized. 2
Labdane-Dial from Aframomum danielli
[0030] 3
Sakurentin from Eupatorium odorantum
[0031] As discussed previously, the parasite can synthesize
ergosterol (Holz, 1985, supra), also known as pro-vitamin D2
(structure shown below) but they require cholesterol which cannot
be synthesized by the organism, and therefore, has to be salvaged
from the host. When leishmania infects a host, within minutes, the
organism localizes to the liver, and it is in the liver that host
ergosterol (provitamin D) is converted to vitamin D.sub.2. The
conversion of ergosterol to cholesterol causes an increase in
Ca.sup.++ ion concentration. It had been reported previously that
the ability of macrophages to kill leishmania is reduced under
increased Ca.sup.++ conditions (Olivier, 1996, Parasitol Today 12,
145-150).
[0032] The structures of the active ingredients in the medicinal
plants, and the fact that the parasites must have to scavenge
cholesterol, made us focus on the cholesterol synthesis pathway as
described in FIG. 1.
[0033] We have found that drugs known to inhibit different parts of
the cholesterol pathway can be, for the first time, used as
antiparasitic agents. This discovery was novel and unexpected and
was the result of putting together several different disparate
pieces of evidence. None of the drugs discussed in this application
were used or suggested for the treatment of leishmania,
trypanosomiasis, or trichomoniasis. It is only after the
elucidation of the chemical structure of the active compounds in
the medicinal plants in addition to inventive activity that the
relationship between the sterol pathway and possible antiparasitic
agents was discovered. Most are human-use, FDA-approved drugs for
alternative medical indications.
[0034] Our initial work focused on the following metabolic steps of
leishmanial steroid metabolism which we have ascertained are
critical for parasite survival: (1) butyric acid as a required
precursor for both fatty acid and sterol synthesis; (2) mevalonic
acid synthesis from acetylCoA; (3) squalene synthesis from
mevalonic acid; (4) ergosterol synthesis from lanosterol; and (5)
sterol (cholestane-analog uptake).
[0035] At each step and in each category of inhibitory compounds,
suitable examples of drugs which may be used as
antiparasitic/antifungal agents are mentioned. However, these
examples are not meant to be limiting, and it is understood that
other suitable drugs, known or to be discovered, which belong in
the categories mentioned can be assayed and used as
antiparasitic/antifungal agents. The assays for testing whether or
not a drug is antiparasitic/antifungal are known, one of which is
described in the Examples below.
[0036] Butyrate Inhibition
[0037] Butyrate is a key fatty acid precursor of acetyl-CoA.
Acetyl-CoA and free fatty acids are critical to eukaryotic cells'
energy production via beta-oxidation. Fatty acids are activated to
acetyl-CoA derivatives, transported into the matrix of the
mitochondria via the carnitine cycle, where they undergo
beta-oxidation (Murray et al., 1988, Harper's Biochemistry, 21st
ed., Appleton and Lange, Publ., Norwalk, Conn.). Beta-oxidation of
fatty acids results in the reduced coenzymes FADH2 and NADH. The
oxidation of 1 mole of FADH2 yields 2 moles ATP, and the oxidation
of 1 mole of NADH yields 3 moles of ATP. From work in our
laboratory, we know that butyrate is a key factor for leishmanial
metabolism. Using .sup.14C-labelled butyrate, we showed that it is
readily taken up and rapidly metabolized to 14CO.sub.2 by
Leishmania spp. (Jackson, et al., 1989, Am J Trop Med Hyg 41,
318-330; Jackson, et al., 1990, Am J Trop Med Hyg 43, 464-480). Any
compound comprising a butyrate inhibitor can be used as an
antiparasitic/antifungal agent. Suitable forms of such compounds
are cefaloglycin and xenbucine. Cefaloglycin reduces oxidation and
uptake of butyrate. Cefaloglycin,
7-(2-amino-2-phenylacetamido)-3-(hydroxymethyl)-8-oxo-5-Thia-1-azabicyclo-
[4.2.0]acetate (ester), chemical registration no. 3577-01-3, or
aminophenylacetamido cephalosporanic acids, are known in the art
and marketed under the name Kafocin.RTM. by Eli Lilly and Co.
Indianapolis, Ind. A process for their production is described in
U.S. Pat. No. 3,422,103 to Wilfred et al., Jan. 14, 1969, herein
incorporated in its entirety. Xenbucin, 2-(4-biphenyl)butyric acid;
alpha-ethyl-[1,1'-bipheny- l]-4-acetic acid, chemical ID no.
959-10-4, described in Brit. 1,168,542 (1969, Maggioni),
preparation described in U.S. Pat. No. 4,542,233 to Piccolo et al.,
September, 1985, marketed under the name Liosol.RTM. by Maggioni
Pharmaceutici, Italy.
[0038] CHOLINE: Choline is the starting material for lipogenesis
via production of acetyl-CoA. Dapsone (4,4'-diaminodiphenyl
sulfone) has been reported active against human leishmaniasis via
choline inhibition (Dogra, 1992, Infection 20, 189-191). This drug
is believed to act via paraminobenzoic (PABA) acid-reversible block
of the folic acid metabolism of parasitic protozoa. It is unlikely
that this is the mechanism by which dapsone functions against
Leishmania.
[0039] Leishmania rely exclusively on salvage mechanisms for purine
synthesis and metabolism. Presumably, a dapsone block of purine
synthesis via prevention of the reduction of folic acid to the
tetrahydro-derivative and, thus, transport of the formyl carbon
into the purine ring (positions 2 & 8 of purine), could not
occur in leishmanial parasites utilizing preformed purines to
synthesize nucleic acids and lacking these de novo synthetic
pathways Likewise, a thymidylate synthetase block is unlikely to
prove fatal, since Leishmania salvage as well as synthesize
pyrimidines.
[0040] A choline inhibitory pathway for antileishmanial activity
(as suggested by Dogra, 1991, Trans R Soc Trop Med Hyg 85, 212-213;
Dogra, 1992, supra) is more likely, although the mechanism of such
inhibition, is a more complex problem to investigate. Dogra (1991,
supra;1992, supra) postulated that dapsone probably acts against
Leishmania by inhibition of choline incorporation into lecithin in
the cell membrane, thus decreasing phospholipid synthesis. It is
the relationship of choline inhibition to other drug-sensitive
lipid metabolic target(s) that we wish to investigate
therapeutically.
[0041] Dapsone has an IC.sub.50 of 600 mM (1.49 mg/ml) in vitro
against Leishmania major promastigotes in a chemically defined
medium. Dapsone inhibition was not reversible by p-aminobenzoate
(PABA) folate or thymidine (Peixoto and Beverley, 1987, Antimicrob
Agents Chemother 31, 1571-1578). Invanetich and Santi (1990a, FASEB
J 4, 1591-1597) noted that: "Antifolates commonly used to treat
microbial infections are poor inhibitors of Leishmania major
dihydrofolate reductase." Peixoto and Beverley (1987, supra)
concluded that "the mode of action of sulfa drugs [dapsone] is not
by the classical route of de novo folate synthesis". These results
with dapsone inhibition are understandable based on previous work
on the folate metabolism of these protozoan parasites.
[0042] Clofazimine,
N,5-Bis(4-chlorophenyl)-3,5-dihydro-3-[(methylethyl)im-
ino]-2-phenazinamine;
3-(p-choroanilino)-10-(p-chlorophenyl)-2,10-dihydro--
2-(isopropylimino)phenazine, chemical registration no. 2030-63-9,
marketed as Lamprene.RTM., an anticancer and antimycobacterial
riminophenazine drug, is active via phospholipase A2-mediated
oxidative and nonoxidative mechanisms (Arunthathi and Satheesh
1997, Lepr Rev 68(3), 233, 241; Ruff et al., 1998, Ann Oncol 9,
217-219: van Rensburg, et al., 1993, Cancer Res 53, 318-323;
Venkastesan, et al., 1997, Lepr Rev 68, 242-246). Antimycobacterial
dose is 50 mg/day or 100 mg on alternate days (Venkastesan, et al.,
1997, supra). Riminophenazine drugs have never been used or
proposed as antileishmanial/antitrypanosomals. Human dose
recommended are 100-200 mg/day, although doses 400 mg-600 mg/day
can be given.
[0043] Other suitable examples of inhibitory compounds include
eldacimibe,
1,3-Dioxane-4,6-dione,5-[[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]ami-
no][[[4-(2,2-dimethylprophyl)phenyl]-methyl]hexylamino]methylene]-2,2-dime-
thyl-;(2) Cyclic
isopropylidene[(3,5-di-tert-butyl-4-hydroxyanilino)[hexyl-
(p-neopentyl-benzyl)amino]methylene]malonate, chemical registration
no. 141993-70-6, marketed as Eldacimibe.RTM. by Wyeth-Ayerst
Laboratories, Philadelphia, Pa., and lecimibide, Urea,
N'-(2,4-difluorophenyl)-N-(5-((4-
,5-diphenyl-1H-imidazol-2-yl)thio-)pentyl)-N-heptyl-, chemical
registration no. 130804-35-2, marketed as Lecimibide.RTM. by Merch
Pharmaceutical Co., Whitehouse Station, N.J.
[0044] Squalene
[0045] Some compounds act indirectly on the Leishmania as steroidal
synthesis regulators: human insulin, human transferrin, and low
density lipoprotein (LDL). Both transferrin and insulin are either
inhibitors or growth stimulants of human and, possibly also,
leishmanial sterol synthesis depending on concentration
(Schroepfer, 1981, Ann Rev Biochem 50, 585-621; Thompson, 1992, The
Regulation of Membrane LiDid Metabolism. CRC Press Ann Arbor, pp
230; Jackson, et al., 1989, supra). Other sterols, synthesized only
by the Leishmania and fungi, may act to regulate the host cells
(monocyte or macrophage) to prevent parasite killing, e.g. by
increasing intracellular Ca.sup.++ level (Oliver, 1996, supra).
Sacchettini and Poulter (1997, Science 277, 1788-1789) noted that
the isoprenoids, or steroidal building blocks, are a remarkably
diverse chemical class comprising over 23,000 individual compounds.
For over 100 years, dating back to traditional medicine, it has
been known many antifungals also sometimes have antiparasitic
properties (reviewed, Steck, 1972, The Chemotherapy of Protozoan
Diseases, Vol II, p 7.61-7.63 and 11.100-110, U.S. Government
Printing Office, Washington, D.C., #O-462-576). Additionally, it
has been known for over 50 years that antifungals such as
amphotericin B, pentamidine, and ketoconazole (Neal, 1987, The
Leishmaniases in Biology and Medicine, Vol II Clinical Aspects and
Control. Academic Press, New York, pp. 793-845) have
antileishmanial activity. Lipid analyses of several Leishmania spp.
revealed that these parasites' membranes contain a high percentage
of ergosterol, a sterol most frequently found in fungi and some
bacteria (Holz, 1985, supra) which presents a basis for common
mechanism of action of antifungal drugs on leishmania. Terbinafine
is recognized as an clinical antifungal and cutaneous antibacterial
(Back, et al., 1992, Brit J Dermatol 126 (Suppl 39), 14-18;
Baudraz-Rosselet et al., 1992, Brit J Dermatol 126 (Suppl 39),
40-46; Finlay, 1992, Brit J Dermatol 126 (Suppl 39), 28-32;
Goodfield, 1992, Brit J Dermatol 126 (Suppl 39), 33-35; Hay and
Stratigos, 1992, Brit J Dermatol 126 (Suppl 39), 1-69; Haroon, et
al., 1992, Brit J Dermatol 126 (Suppl 39), 47-50; Hull and Vismer,
1992, Brit J Dermatol 126 (Suppl 39), 51-55; Kovarik, et al., 1992,
Brit J Dermatol 126 (Suppl 39), 8-13; Nolting and Brautigam, 1992,
Brit J Dermatol 126 (Suppl 39), 56-60; Roberts, 1992, Brit J
Denmatol 126 (Suppl 39), 23-27; Ryder, 1992, Biochem J 230,
765-770; Van der Schroeff, et al., 1992, Brit J Dermatol 126 (Suppl
39), 36-39; Villars and Jones, 1992, Brit J Dermatol 126 (Suppl
39), 61-69).
[0046] Recent antiparasite investigations of known antifungals have
primarily involved the combination of known antileishmanials with
one or more newer antifungals, the latter to include the squalene
oxidase inhibitor, terbinafine. The antifungal terbinafine has
shown preliminary antitrypanosomal activity in vitro and in primary
rodent drug screening systems against Trypansoma cruzi, the
etiologic agent of Chagas' disease (Urbina et al., 1996, Science
273, 969-971) and Leishmania mexicana, 2 cutaneous leishmanial
subspecies (Goad et al., 1985, Biochem Pharmacol 34, 3785-3788;
Berman and Gallalee, 1987, J Parasitol 73, 671-673).
[0047] Complex structure activity relationship (SAR) studies of
synthetic and natural product (biologically derived) squalene
synthetase and squalene oxidase inhibitors have shown several such
compounds have in vitro and in vivo activities having human
hypocholesteremic potential. Abe and collegues (1994, supra)
reviewed SAR data from 284 squalene synthesis inhibitors. Selected
data from a few of the best hypocholesteric candidates (from Abe,
et al, 1994 supra) follow.
[0048] Suitable examples of Squalene Synthetase inhibitors
include:
[0049] 1. Thioether analog of 2,3-oxidosqualene (Abe, et al, 1994,
supra; Zheng, et al, 1995, J Am Chem Soc 117, 670-680) ICC.sub.50
0.0023 uM
[0050] 2. 29-methylidene-2,3-oxidosqualene, an irreversible
inhibitor of oxidosqualene cyclase (Abe, et al, 1994, supra; Xiao
and Prestwich, 1991, J Am Chem Soc 113, 9673-9674)
[0051] 3. Ether analog of farnesyl diphosphate (IC.sub.50 0.05 uM,
Abe, et al, 1994, supra)
[0052] 4. Farnesyl bisphosphonate (no oral activity, IC.sub.50
0.00027 uM, Abe, et al, 1994, supra)
[0053] 5. Natural product from Phoma sp. C2932, Squalestatins 1,2,3
(IC.sub.50 15.2, 15.1, 5.9 nM, respectively, Abe, et al, 1994,
supra)
[0054] 6. Natural products from ATCC 20986, Sporormilla intermedia,
and Leptodontium elatius: Zaragozic acid A,B,C, IC.sub.50 78, 29,
45 pM , respectively (Abe, et al, 1994, supra)
[0055] 7. CP-225,917 (Pfizer) and CP-263,114 (Pfizer), both
compounds inhibit squalene synthase and farnesylprotein transferase
(Borman, 1999, Chemical and Engineeing News Jun. 7, 1999, 8-9;
Service, 1999, Science 284, 1598-1599; Dabrah et al., 1997, J
Antibiot 50, 1-7)
[0056] Suitable examples of inhibitors of Squalene Oxidase
include:
[0057] 1. Naftifine, 1-Naphthalenemethanamine,
N-methyl-N-(3-phenyl-2-prop- enyl)-(E), chemical registration no.
65472-88-0, marketed as an antifungal under Exoderil.RTM. or
Naftin.RTM., and described in a patent to Berney on Aug. 4, 1981,
U.S. Pat. No. 4,282,251. IC.sub.50 0.93 uM (Abe et al, 1994, supra;
Georgopoulis et al., 1981, Antimicrob Agents Chemother 19, 386-389;
Paltauf et al., 1982, Biochim Biophys Acta 712, 268-273; Petranyi
et al., 1984, Science 224, 1239-1241; Ryder, 1984, In Nombel C.
(ed.) Microbial Cell Wall Synthesis and Autolysis, Elsevier, N.Y.,
pp 313-321)
[0058] 2. Terbinafine, 1-Naphthalenemethanamine,
N-(6,6-dimethyl-2-hepten-- 4-ynyl)-N-methyl-, (E)-, an antimycotic
allylamine, chemical registration no. 91161-71-6, or turbinefine
hydrochloride, chemical registration no. 78628-80-5. Turbinafine is
marketed as Lamisil.RTM., and its preparation is described in Eur
Patent Appl. no. 24,587 to A. Stutz, 1981. Terbinafine has been
shown to have activity against Leishmania species in vitro and in
animal and human clinical trials (Abe, et al, 1994, supra; Bahamdan
et al., 1997, Int J Dermatol 36, 59-60; Ellenberger and Beverley,
1989, J Biol Chem 264, 15094-15103; Goad, et al., 1985, Biochem
Pharmacol 34, 3785-3788; Gonzales-Ruperez et al., 1997, Dermatology
194, 85-86; Rangel et al., 1996, Antimicrob Agents Chemother 40,
2785-2791; Urbina 1997, Parasitology 114 Suppl S91-S99;
Vannier-Santos et al., 1995, J Eukaryot Microbiol 42, 337-346).
[0059] 3. Butenafine,
N-(p-tert-Butylbenzyl)-N-methyl-1-naphthalenemethyla- mine, a
benzyl amine antifungal, chemical registration no. 101828-21-1, or
butenafine hydrochloride, chemical registration no. 101827-46-7,
marketed as Mentax.RTM. by Penederm Inc. Foster City, Calif.
Preparation is described in U.S. Pat. No. 4,822,822 to Arita et al.
on Apr. 18, 1989.
[0060] 4. SDZ 87-469 (Georgopapadakou et al., 1992, Antimicrob
Agents Chemother 36, 1779-1781, and references cited therein; Ryder
and Frank, 19992, J Med Vet Mycol 30, 452-460) IC.sub.50 0.011 uM
(Abe, et al, 1994, supra)
[0061] 5. NB-598, (Matzno et al., 1997, J lipid Res 38, 1639-1648
and references cited therein) IC.sub.50 0.75 nM (Abe, et al, 1994,
supra)
[0062] 6. TMD, 4,4,10beta-trimethyl-trans-decal-3beta-ol (Abe, et
al, 1994, supra; Nelson et al, 1978, J Am Chem Soc 100,
4900-4902)
[0063] HMGCOA, 3-hydroxy-3-methylglutaryl CoA Reductase
Inhibitors
[0064] Mevalonic acid, a precursor to human sterols and steroids;
and in plants, to hormones and carotenoids, is available to
Leishmania both via the host human monocyte or macrophage; and
within the sandfly vector, in the bloodmeal and plant juices
essential to sustain the fly (Leclercq, 1969, Entomological
Parasitology. Pergamon Press, New York, pp 158; Beytia and Porter,
1976, Ann Rev Biochem 45, 112-142; Thimann, 1977, Hormone Action in
the Life of Plants. University of Massachusetts Press, Amherst, pp
448; Caspi, 1984, Tetrahedron 42, 3-50). Most sandfly species known
to transmit Leishmania, require not only blood but also plant
fluids to maintain proper hydration for survival. Avoiding lethal
ultraviolet rays of sunlight, sandflies rest in moist shady areas
during the day to emerge-in the evening to feed. Plants,
particularly those in the tropical climates where sandflies are
most numerous, tend to lose water in the intense heat of the
afternoon. The plant hormone responsible for closing leaf stomata
to prevent plant dehydration, abscisic acid, is made in response to
water loss. Abscisic acid is known to increase 200-fold in a
dehydrated plant. Three mevalonic acid molecules are required to
produce one molecule of abcissic acid. As more abscisic acid is
required in the heat of the day, so is its precursor, mevalonic
acid. Sandflies feed at twilight when plant dehydration, abscisic
acid, and melvalonic acid would be expected to be near peak daily
level in tropical plants.
[0065] In animals and humans, mevalonic acid is also an important
precursor to sterol and steroid synthesis, so would likewise be
available to leishmanial amastigotes inhabiting the monocytes,
macrophages and hepatic cells. On the basis of host and vector
physiology alone, mevalonic acid appeared to be implicated as an
important precursor molecule for leishmanial sterolgenesis.
[0066] To investigate this, we used .sup.14C-mevalonic acid to
determine rate of incorporation by Leishmania (1.0 ng/hr/108
parasites at 25.degree. C., using the respirometric assay. We also
looked at mevalonic acid catabolism and found mevalonate is
sparingly metabolized to CO2 (less than 1/25 the rate of aspartic
acid metabolism, a most rapidly catabolized amino acid, Jackson, et
al., 1989, Am J Trop Med Hyg 41, 318-330) even when the
promastigotes are maintained under starvation condition for 30
minutes. When mevalonate was added as a nutritional supplement the
parasites grew profusely but less rapidly than parallel
unsupplemented control cultures. (The amount mevalonate added to in
vitro cultures was determined based on incorporation rate relative
to aspartic acid, and this may have resulted in too high an
estimated mevalonic acid concentration.) However, mevalonate-fed
cultures remained in logarithmic phase growth 2-fold longer (>10
days) than parallel unsupplemented cultures (which ended log phase
growth at 4-5 days of culture). Given these preliminary
observations: it appears Leishmania (a) incorporate mevalonic acid
readily from their environment; (b) catabolism is spared even under
starvation conditions; and (c) mevalonic acid can act as a
nutritional supplement in vitro.
[0067] Three-hydroxy-3-methylglutaryl CoA reductase is a protein of
the endoplasmic reticulum whose concentration is determined by
rates of cholesterol synthesis. HMG-COA reductase catalyzes the
reductive deacylation of HMG-COA to mevalonate by two molecules of
NADPH. In most tissues this is considered the first committed step
in sterol/isoprenoid biosynthesis. In most biologic systems
studied, this reaction is the rate-limiting step for sterol
biosynthesis (Danielsson and Sjovall, 1985). Most widely used
hypercholesteremic drugs have their mode of action at this
irreversible synthetic step catalyzed by 3-hydroxy-3-methylglutaryl
CoA reductase (HMCoAR).
[0068] HMG-COA reductase inhibitors lower plasma total cholesterol,
low density lipoprotein (LDL), and B apolipoprotein in humans as
the result of decreased cholesterol synthesis and enhanced removal
of LDLs via the LDL receptor pathway in hepatocytes (Hoeg and
Brewer, 1987; Tolbert, 1987).
[0069] No HMG-COA reductase inhibitor has ever been used or
previously tested as an antileishmanial or for South and Central
American Trypansoma rangeli. There are two references to
anti-Trypanosoma (Schizotrypanum) cruzi, South and Central American
trypanosome species, in vitro (Florin-Christensen, et al., 1990;
Urbina, et al., 1993) and in vivo mouse testing of mevinolin
(Lovastatin.RTM.) testing, alone and in combination with
ketoconazole and terbinafine (Urbina, et al., 1993). Coppens and
colleagues (1995; and, Coppens and Courtoy, 1995) showed that the
enzyme inhibitor, synvinolin (simvastatin or Zocor.RTM.),
potentiates growth inhibition of Trypanosoma brucei in the presence
of drugs interfering with the exogenous supply of cholesterol; and
conversely, growth inhibition by synvinolin can be reversed by LDL,
mevalonate, squalene or cholesterol.
[0070] All 3-hydroxy-3-methylglutaryl CoA reductase inhibitors, or
vastatins, are not a chemical class effect but vary widely between
HMGCoA reductase inhibitors (Bocan, et al., 1994; Haruyama, et al.,
1986; Kempen, et al., 1991; Nakaya, et al., 1986; Serizawa, et al.,
1983; Tsujita, et al., 1986; Yoshino, et al., 1986)(e.g.
atorvastatin, CI981; PD134965; pravastatin, CS-514, Eptastatin, SQ
31000; BMY22089; simvastatin, Synvinolin, MK-733; monacolin K,
MB-530B; mevinolin, lovastatin; mevastatin, ML-236B, Compactin) do
not have the same efficacy for preventing atherosclerotic lesions,
inhibition of cholesterol synthesis in target tissue(s), reducing
cellular accumulation of free and/or esterified cholesterol,
degradation of LDL, or synthesis of phosphotidylcholine and
sphingomyelin.
[0071] This observation may be due, in part to the chemical design
of various vastatins, for example, pravastatin differs from other
HMG-COA reductase inhibitors in two aspects. In pravastatin, the
6-position on the decalin ring is occupied by a hydroxyl group,
whereas, in lovastatin and simvastatin, this same position is
occupied by a methyl group. This difference in structure is
responsible for the different physiochemical properties of these
drugs and confers on pravastatin its hydrophilic characteristics.
Lovastatin and simvastatin are hydrophobic and designed with the
objective of obtaining high levels of hepatoselectivity (Keidar, et
al., 1994; Sirtori, 1993). Pravastatin is administered as a sodium
salt of an open acid and is the active inhibitor of HMG-CoA
reductase; lovastatin and simvastatin are prodrugs and are given as
inactive lactones that, following oral administration, are
hydrolyzed to an active inhibitor" (Keidar, et al., 1994).
Pravastatin is manufactured by Bristol-Myers Squibb; Merck
manufacturers lovastatin and simvastatin (Zurer, 1997); and Sanyo,
eptastatin (Yoshino, et al., 1986).
[0072] Suitable HMG-CoA reductase inhibitors include:
[0073] 1) Pravastatin, [1S-(1-alpha(beta-S*,delta-S*),
2-alpha,6-alpha,8-beta(R*),
8a-alpha]]-1,2,6,7,8,8a-hexahydro-2-methyl-8--
(2-methyl-1-oxobutoxy)-beta,delta,6-trihydroxy-1-Naphthaleneheptanoic
acid monosodium salt, a highly selective cholesterol synthesis
inhibitor of hepatic, intestinal cells (ileum), and in
monocyte-derived macrophages (Keidar, et al., 1994). Pravastatin,
chemical registration no. 81093-37-0, marketed as Pravachol.RTM. by
Bristol-Myers Squib, Wallingford, Conn. or as Eptastatin from
Sanyo, as well as others. The preparation of pravastatin is
described in U.S. Pat. No. 4,346,227 to Terahara et al., August,
1982. When humans were given a dose of 40 mg/day for 8 weeks,
pravastatin resulted in a dose-dependent inhibition of macrophage
cholesterol synthesis; LDL increased 119% with 0.1 mg/ml
pravastatin; <or =0.19 mg/ml increased cholesterol
esterification; >0.19 mg/ml inhibited cholesterol
esterification; pravastatin inhibited cholesterol synthesis 55-62%
and increased LDL degradation by 57% (Keidar, et al., 1994).
[0074] 2) Simvastatin, [1S-[1-alpha(beta-S*,delta-S*),
2-alpha,6-alpha,8-beta(R*), 8a-alpha]]2,2-dimethylbutanoic acid
1,2,3,7,8,8a-hexahydro-3,7-dimethyl-8-[2-(tetrahydro-4-hydroxy-6-oxo-2H-p-
yran-2-vl)ethyl]-1-naphthalenyl ester, a competitive inhibitor of
HMG-COA reductase, chemical registration no. 79902-63-9, marketed
in several forms, e.g. Zocor.RTM. from Merck & Co., Whitehouse
Station, N.J., preparation described in U.S. Pat. No. 4,444784 to
Hoffman et al. April, 1984. In a longterm study of simvastatin
(3-5.4 years) at doses 0.5 of pravastatin and 0.125 of fluvastatin,
simvastatin (at 10 to 40 mg/day doses) lowered serum cholesterol
from baseline 20-40%; lowered low density lipoprotein cholesterol
35-45%; and reduced triglycerides 10-20% (Plosker GL, McTavish D,
1995).
[0075] 3) Fluvastatin, 6-Heptenoic acid,
3,5-dihydro-7-[3-(4-fluorophenyl)-
-1-(1-methylethyl)-1H-indol-2-yl-]-[R*, S*(E)]-, (+-)-, chemical
registration nos. 93957-55-2 and 93957-54-1, marketed as
Lescol.RTM. from Sandoz, East Hanover, N.J., described in U.S. Pat.
No. 4,739,073, 1984. Review of Pharmacology and therapeutics use,
Levy et al., 1993, Circulation 87, Suppl III-45 to III-53.
[0076] 4) Atorvastatin, 1H-Pyrrole-1-heptanoic acid,
2-(4-fluorophenyl)-beta,delta-dihydroxy-5-(1-methylethyl)-3-pheny-1-4-[(p-
henylamino)carbonyl)-, [R-(R*,R*)]-, chemical registration nos.
134523-00-5 and 11086248-1, described in U.S. Pat. No. 5,273,995 to
Roth, December 1993, marketed by Warner-Lambert, Morris Plains,
N.J.
[0077] 5) Cerivastatin,
7-[4-(4-fluorophenyl)-5-(methoxymethyl)-2,6-bis(1--
methylethyl)-3-pyridinyl]-3,5-dihydroxy-, monosodium salt,
[S-(R*,S*-(E))]]-, cerivastatin sodium, chemical registration no
143201-11-0, marketed as Baycol.RTM. from Bayer Corp. West Haven,
Conn.
[0078] 6) Crilvastatin, L-Proline, 5-oxo-,
3,3,5-trimethylcyclohexyl ester, chemical registration no.
120551-59-9, available from Laboratoire Pan Medica, France.
[0079] 7) Dalvastatin,
2H-Pyran-2-one,tetrahydro-6-[2-(2-(4-fluoro-3-methy-
lphenyl)-4,4,6,6-tetramethyl--1-cyclohexen-1-yl]ethenyl]-4-hydroxy-,
[4R-(4-alpha,6-beta(E)]]-, chemical registration nos. 135910-20-2,
132100-551, available from Rhone-Poulenc Rore Pharmaceuticals, Inc.
Collegeville, Pa.
[0080] 8) Lovastatin, Butanoic acid,
2-methyl-,1,2,3,7,8,8a-hexahydro-3,7-- dimethyl
-8-[2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl]-1-naphthal-
enyl ester,[1S[1alpha(R*), 3alpha,7beta,8beta(2S*,4S*), 8abeta]]-,
chemical registration no. 75330-75-5, marketed in several forms,
e.g. Mevacor.RTM. from Merck & Co., Inc. Whitehouse, N.J.,
described in U.S. Pat. No. 4,231,938 to Monaghan et al., November
1980, and G. S. Brenner et al., in Analytical Profiles of Drug
Stubstances and Excipients, vol 21, H.g. Brittain, Ed. (Academic
Press, San Diego, 1992) pp 277-305.
[0081] 9) Mevastatin, Butanoic acid,
2-methyl-,1,2,3,7,8,8a-hexahydro-7-me-
thyl-8-[2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl]-1-naphthalenyl
ester,[lS-[1-alpha(R*), 7-beta,8-beta(2S*,4S*), 8a-beta]]-,
chemical registration no. 73573-88-3, marketed is several forms,
e.g. Compactin.RTM. from from Merck & Co., Inc. Whitehouse,
N.J., and described in U.S. Pat. No. 3,983,140 to Endo et al.,
September 1976 and reviewed in Endo, 1985, J Med Chem 28,
401-405.
[0082] Nonlipid Related Effects of Certain HMGCoA Reductase
Inhibitors
[0083] Fluvastatin, simvastatin, and lovastatin (but not
pravastatin) locally inhibit isoprenoid biosynthesis resulting in
the following antiatherosclerotic effects on the arterial wall: a)
inhibition of smooth muscle cell migration and proliferation
(reversed by mevalonate); b) fluvastatin and simvastatin also
inhibit cholesterol esterification and deposition induced by
acetylated LDL in cultured macrophages (Corsini, et al., 1996).
Simvastatin and lovastatin also reduce the rate of DNA synthesis
and proliferation of a wide variety of cell types in vitro, by
inducing a cell cycle arrest in G1 (Addeo, et al., 1996; Wilcken,
et al., 1997). This effect of simvastatin and lovastatin on DNA
synthesis arrest is antagonized by estrogen (Addeo, et al.,
1996).
[0084] Inhibitors of Cholesterol Bile Acids Recycling:
7-alpha-hyroxylase and relationship to HMG-CoA reductase
[0085] Cholesterol 7-alpha-hyroxylase and HMG-CoA reductase are
located near each other on the endoplasmic reticulum. Newly
synthesized cholesterol seems to be the preferred substrate for
cholesterol 7-alpha-hyroxylase and its diurnal rise correlates with
rise in enzyme synthesis. This enzyme is intricately linked with
sterol synthesis and it's regulation (Danielsson and Sjovall,
1985). Therefore, it seemed logical to assume that certain
inhibitors of cholesterol-bile acid recycling from the intestine
may have cholesterol lowering effects that would also act to lower
host tissue cholesterol available to parasites.
[0086] However, although bile acid binding drugs have not proven,
to date, to be active used alone against parasites, these compounds
may enhance HMG-COA reductase inhibitor activity as demonstrated by
Hoogerbrugge, et al., 1990; Kuroda, et al., 1992; McTavish and
Sorkin, 1991; and, Wiklund, et al., 1993. This combination of an
HMG-CoA reductase inhibitor plus a bile acid binding drug is likely
be more potent for antiparasitic therapy than any single HMGCoA
reductase inhibitor alone because of known enhanced
anticholesteremic properties of the two drug types when
administered together over either drug type given alone.
[0087] Medical concern that hypocholesteremics based on HMG-CoA
reductase inhibition may result in untoward effects on nontarget
tissues due to longterm physiologic consequences of depletion of
mevalonate-derived isoprenoids led to examination of cholesterol
inhibition further down the synthetic path, at squalene
synthesis.
[0088] Cytochrome P450 Enzyme Inhibitors: 14Alpha-Demethylase
Inhibition and Delta 24(25) Sterol Methyltransferase Inhibitors
[0089] The cytochrome P450 enzymes are a family of iron-containing
hemoproteins. The P450 enzymes are generally divided based on
structure and function. Those involved in steroidogenesis, the
CYP11, CYP17, CYP19, CYP21 and CYP27 subfamilies; and in the
metabolism of cholesterol and bile acids, the CYP7 and CYP51
subfamilies exhibit a high degree of regio- and stereospecificity
(Coon, et al., 1992; Mason and Hutt, 1997; Nebert, et al., 1991).
Coincidentally, in evolutionary terms, those cytochrome P450
enzymes involved in steroidogenesis are also the oldest mammalian
P450's. Therefore, shared P450 steroidal enzymes are the most
likely to be common to both humans and more primitive fungal or
protozoan parasites infecting humans. Therefore, drugs known to
specifically inhibit these P450 steroidal enzymes may also inhibit
similar P450 enzymes of older, more primitive organisms.
[0090] Imidazole drugs, using ketoconazole as an example, may then
have antifungal/antiparasitic action for two reasons: (1) Direct
action on parasite P450 steroidogenic enzymes reduces parasite de
novo sterol synthesis, particularly fungal and protozoal-specific
ergosterol synthesis via 14alpha-demethylase inhibition of
lanosterol conversion to ergosterol. (2) Also, indirectly because
the human host intracellular or blood environment where the
parasites must obtain cholesterol by "salvage" is likewise depleted
of this second sterol required for leishmanial and trypanosomal
survival.
[0091] Imidazoles can inhibit transformation of lanosterol to
either Ergosterol or Cholesterol (14alpha-) Imidazoles are
typically considered "antifungals" for use in treatment of both
superficial and systemic fungal infections (Heel, et al., 1982,
Drugs 23, 1-28). However, various other physiologic-drug effects
with rising doses have resulted in use of these compounds for
nonfungal indications. Examples of imidazoles include:
ketoconazole, clotrimazole, aminoglutethimide, and etomidate. Doses
and pharmacokinetics for imidazoles have been reviewed by Heel et
al., 1982, supra.
[0092] The antifungal compound, ketoconazole, is believed to
inhibit cholesterol biosynthesis via inhibition of the microsomal
P-450 enzyme 14alpha-demethylase. Additional known drug activities
affecting steroidogenesis of imidazoles in general and ketoconazol
in particular include: (1) at therapeutic doses (200-600 mg/day)
ketoconazole blocks testosterone synthesis in men (Feldman, 1986;
Pont, et al., 1982) and at high dose regimens caused substantial
inhibition of testicular and adrenal steroidogenesis (Feldman,
1986); (2) ketoconazole blocks 11beta-hydrolase and cholesterol
side-chain cleavage for the adrenal steroidogenic pathway (Feldman,
1986); (3) ketoconazole inhibits renal 25-hydroxyvitamin
D-24-hydroxyase (Vitamin D, an intracellular Ca.sup.++ regulator)
(Feldman, 1986).
[0093] The actions of ketoconazole (as a representative imidazole)
decreased human patient plasma cholesterol between 27% (at 1200
mg/day) to 15% (at 200 mg/day) from pretreatment baseline (Feldman,
1986). Ketoconazole and two other related 24(25) sterol
methyltransferase inhibitors were shown by Urbina, et al. (1995) to
elucidate that 24-alkyl sterols are essential growth factors for
Trypanosoma cruzi and that the preferred substrate of the delta
24(25) sterol methyltransferase in this organism is zymosterol.
[0094] Miscellaneous Hypocholesteremics
[0095] 1) BERBERINES: The exception to these lipid cogener natural
antiparasitics are several natural and synthetic berberine/berbine
analogs (U.S. Pat. No. 5,290,553, to Iwu, et al., 1994). Berberine
extracted from Coptis chinensis, lowered serum cholesterol level of
mice fed a high cholesterol diet (Chen and Xie, 1986) and is a
known hypocholesteremic. These natural and synthetic
berberine/berbine analogs have been found to have potent
antimalarial, antitrypanosomal, and antileishmanial properties
(U.S. Pat. No. 5,290,553, to Iwu, et al., 1994)
[0096] 2) BETA-CAROTENE AND LYCOPENE are moderate
hypocholesteremics. Fuhrman, et al. (1997, Biochem Biophys Res
Commun 233, 658-662) reported a 14% decrease in plasma LDL
cholesterol, in humans given a dose of 60 mg/day tomato lycopene
for 3 months. In vitro, J-774 A.1 macrophages' cholesterol
synthesis was inhibited 63% or 73% from acetate, but not from
mevalonate, following treatement with 10 uM beta-carotene or
lycopene, respectively (Fuhrman, et al., 1997, supra).
[0097] 3) ANTICANCER COMPOUNDS: In some cases, anticancer agents
act because sterol synthesis in proliferating cells is ususally
controlled by sterols that are produced intracellularly and is,
independent of extracellular cholesterol (Danielsson and Sjovall,
1985, Sterols and Bile Acids. Elsevier, N.Y.). A linkage has been
shown between de novo cholesterol synthesis and is required for
completion of the cell cycle (Bottomley, et al., 1980, FEBS Lett
119, 261-264). It would be expected that such anticancer agents
(e.g. estrogen/testosterone agonists/antagonists) would have some
antiparasitic properties either by virtue of lowering the
cholesterol of the parasites' enviroment within the mammalian host
(including man) or by direct inhibitory action on the
sterol/cholesterol synthetic pathway of the parasites. It is well
known (see above discussion) that while parasite and mammalian
sterol metabolic pathways differ in some basic fundamental steps,
these pathways for sterol production and incorporation share many
common substrates, enzyme cofactors, and result in the same
products. Thus, it is not unreasonable to assume that an anticancer
compound having a known mode-of-action targeting a pathway common
to both parasites and mammals (including man) would have
fundamental and significant antiparasite properties.
[0098] One example is ketoconazole, which at moderate (200-600
mg/day) or high dose regimens inhibits both testicular and adrenal
steroidogenesis (Feldman, 1986, Endocrine Rev 7, 409-420). Examples
include: ketoconazole, clotrimazole, aminoglutethimide, and
etomidate. At 400 mg/3.times./day ketoconazole, prostate cancer
subjects showed clinical improvement with few and minor side
effects (Feldman, 1986, supra; Singh et al., 1995, J Assoc
Physicians India 43, 319-320; Larbi et al 1995, Am J Trop Med Hyg
52, 166-168; Trachtenberg, 1984, J Urol 132, 61; Trachtenberg, and
Pont, 1984, Lancet 2, 433).
[0099] An second suitable example is tamoxifen,
(Z)-2-[4-(1,2-Diphenyl-1-b- utenyl)phenoxy]-N,N-dimethylethanamine,
(chemical registration no. 54965-24-1 and 10540-29-1, preparation
described in U.S. Pat. No. 4,536,516 to Harper et al., August
1985), best known as a nonsteroidal estrogen agonist for breast
cancer adjuvant therapy (Bryant and Dere, 1998, Proc Soc Exp Biol
Med 217, 45-52; Major et al., 1998, Orv Hetil 139, 121-124; Muller
et al., 1998, Cancer Res 58, 263-267). Among tamoxifen's known
consequences is that it results in lowering of sterol synthesis and
cholesterol levels in many body tissues, including significant
decreases in total serum and low density lipoprotein (LDL)
cholesterol levels, increase in high density lipoprotein subclass 2
cholesterol, and increase in apolipoprotein A-I, a decrease in
apolipoprotein B, and a reduction in serum concentration of
lipoprotein (a) in humans (Chang et al., 1996, Ann Oncol 7,
671-675; Elisaf et al., 1996, Anticancer Res 16, 2725-2728; Morales
et al., 1996, Breast Cancer Res Treat 40, 265-270; Wasan et al.,
1997, J Pharm Sci 86, 876-879), and Wistar rats (Vinitha et al.,
1997, Mol Cell Biochem 168, 13-19). These effects on cholesterol
may be due to a direct inhibition of delta-8-isomerase (see Gylling
et al., 1995, J Clin Oncol 13, 2900-2905). A known side-effect
during high-dose therapy (similar to central nervous system toxicty
of antiestrogens of the clomiphene type) is cognitive impairment in
32% of patients, and 17% of standard-dose patients, compared to 9%
of control patients (van Dam et al., 1998, J Natl Cancer Inst 90,
210-218).
[0100] A third example is the estrogen antagonist, Raloxifene,
Methanone,
[6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thien-3-yl][4-[2-(1-piperidinyl)eth-
oxy]phenyl]-, (chemical registration no. 84449-90-1) which inhibits
both copper mediated LDL oxidation as well as the cellular
modification of LDL by murine peritoneal macrophages. Raloxifene is
a more potent inhibitor of LDL oxidation than 17-beta-estradiol and
(in rats) lowered cholesterol levels below control values within 4
days after initiation of treatment (Bryant and Dere, 1998, supra;
Frolik et al., 1996, Bone 18, 621-627; Zuckerman and Bryan, 1996,
Atherosclerosis 126, 65-75). Another estrogen antagonist,
exemestane, 6-methyleneandrosta-1,4-diene-3,17-dione, chemical
registration no. 107868-30-4, an irreversible inhibitor of
steroidal aromatase, reduces total and HDL cholesterol and total
triglyceride.
[0101] A fourth example is the antiestrogen, clomiphene citrate
(Clomid.RTM., Prepn: Allen et al., U.S. Pat. No. 2,914,563 in 1959
to Merrell); droloxifene/droloxifene citrate (Klinge Pharma,
Germany) and Zuclomiphene (=Transclomiphene, Marion Merrell Dow)
which are also know to have hypocholesteremic properties via
inhibition of cholesterol biosynthesis (Ke et al., 1997, Bone 20,
31-39; Ramsey and Fredericks, 1977, Biochem Pharmacol 26,
1161-1167). Droloxifene was reported to reduce total serum
cholesterol 65-70% compared to controls in rats (Ke et al., 1995,
Bone 17, 491-496). Similarly, toremifene (and tamoxifen) are
reported to inhibit the conversion of delta-8-cholesterol to
lathosterol so that total and LDL cholesterol levels are lowered by
downregulation of cholesterol synthesis. Thus, inhibition of the
delta-8-isomerase may be the major hypolipidemic effect of these
agents (Gylling, et al., 1995, supra).
[0102] Many antiestrogens seem to work because estrogen is known to
elevate plasma cholesterol concentration (Klimis-Tavantzis et al.,
1983, J Nutr 113, 320-327); However, the disadvantage is that these
also seem to lower cholesterol biosynthesis in the central nervous
system and neurotoxic effects are known for many antiestrogens
including the clomiphe
[0103] ne and derivatives (Ramsey 1978, Biochem Pharmacol 27,
1637-1640).
[0104] Other possible antiparasitic/antifungal compounds
include:
[0105] Thyroid hormone antagonists, suitable examples include
dextrothyroxine,
D-4-(4-Hydroxy-3,5-diiodophenoxy)-3,5-diiodobenzylalanin- e;
0-(4-hydroxy-3,5-diiodophenyl)-3,5-diiodo-, or D-thyroxine,
chemical registration no. 51-49-0, and dextrothyroxine sodium,
chemical registration no. 7054-08-2 and 137-53-1, marketed as
Choloxin.RTM. by Knoll Pharmaceutical Co. Mount Olive, N.J., U.S.
Pat. No. 2,889,363 to Ginger on June 1959.
[0106] Antihyperlipoproteinemic agents which inhibit cholesterol
reabsorption as bile acids. Suitable examples include
cholestyramine resin, chemical registration no. 58391-37-0 or
11041-12-6 (Ast and Frishman, 1990, J Clin Pharmacol 30, 99-106),
marketed in several forms, e.g. Questran.RTM. from Bristol-Myers
Squib, Wallingford, Conn. In the same category is colestipol,
chemical registration no. 50925-79-6 or colestipol hydrochloride,
1,2-Ethanediamine,N-(2-aminoethyl)-N'-[2-[(2-am-
inoethyl)amino]ethyl]-, polymer with (chloromethyl)oxirane,
chemical registration no. 37296-80-3, preparation described in U.S.
Pat. No. 3,803,237 to Lednicer et al., April 1974, reviewed in Heel
et al., 1980, Drugs 19, 161-180, and marketed as Cholestid.RTM.
from Pharmacia and Upjohn, Inc. Kalamazoo, Mich.
[0107] Antihyperlipoproteinemics, suitable examples include:
[0108] clofibrate, Propanoic acid, 2-(4-chlorophenoxy)-2-methyl-,
ethyl ester, chemical registration no. 637-07-0, described in
Hassan and Elazzouny, 1982, Anal Profiles Drug Subs 11, 197-224,
marketed in several forms, e.g. Atromid-S.RTM. from Wyeth-Ayerst,
Philadelphia, Pa.
[0109] Antihyperlipoproteinemics which inhibit synthesis of VLDL,
possibly by inhibiting synthesis of ApoB-100), for example,
Gemfibrozil, Pentanoic acid, 5-(2,5-dimethylphenoxy)-2,2-dimethyl-;
Valeric acid, 2,2-dimethyl-5-(2,5-xylyloxy)-, chemical registration
no. 25812-30-0, preparation described in U.S. Pat. No. 3,674,836 to
Creger on July, 1972, marketed in several forms, e.g. Lopid.RTM.
from Parke-Davis, Morris Plains, N.J.
[0110] Antihyperlipoproteinemics which inhibit synthesis of
cholesterol and increase fecal excretion of bile acids, and may
decrease plasma HDL levels, e.g. Probucol, Acetone,
bis(3,5-di-tert-butyl-4-hydroxyphenyl) mercaptole;
4,4'-[(1-methylethylidene)bis(thio)]bis[2,6-bis(1,1-dimethyle-
thyl)phenol], chemical registration no. 23288-49-5, preparation
described in U.S. Pat. No. 3,576,883 to Neuworth, M. B. on April,
1971, and its use as a cholesterol-lowering agent in U.S. Pat. No.
3,862,332 to Barnhart et al., on January 1975, marketed in several
forms, e.g. Lorelco.RTM., by Hoechst Marion Roussel, Inc. Kansas
City, Mo.
[0111] Antihyperlipoproteinemics which inhibit cholesterol lumenal
absorption resulting in reduced serum LDL and serum cholesterol
(Morehouse et al., 1999, J Lipid Res 40, 464-474. For example,
Tiqueside, beta-D-Glucopyranoside,
(3beta,5alpha,25R)-spirostan-3-yl 4-O-beta-D-glucopyranosyl-
chemical registration no. 99759-19-0, and Pamaqueside
Spirostan-1]-one,3-[(4-O-beta-D-glucopyranosyl-beta-D-glucopy-
ranosyl)oxy]-, (3beta,5alpha,25R)--; (2)
11-Oxo-(25R)-5alpha-spirostan-3be- ta-yl
4-O-beta-D-glucopyranosyl-beta-D-glucopyranoside, chemical
registration no. 150332-35-7, both available from Pfizer
Laboratories, New York, N.Y.
[0112] Inhibitors of type II fatty acid synthesis such as
cerulenin, 2,3-Epoxy-4-oxo-7,10-dodecadionamide. Antifungal
antibiotic isolated from several species, including Acremonium
(Cephalosporium), Acrocylindrum, and Helicoceras. It inhibits the
biosynthesis of several lipids by interfering with enzyme function,
chemical registration no. 17397-89-6, preparation described in
Boeckman and Thomas, 1977, (J Am Chem Soc 99, 2805).
[0113] Antineoplastic agents, suitable examples including
Ifosfamide,
N,3-bis(2-chloroethyl)tetrahydro-2H-1,3,2-Oxazaphosphorin-2-amine,
2-oxide;
3-(2-chloroethyl)-2-[(2-chloroethyl)amino]tetrahydro-1,3,2-Oxaza-
phosphorine, 2-oxide, chemical registration no. 3778-73-2,
preparation described in U.S. Pat. No. 3732,340 to Arnold et al. in
May, 1973, reviewed in Schoenike and Dana, 1990, Clin Pharm
179-191, marketed in several forms, e.g. Ifex.RTM. from
Bristol-Meyers Oncology Division, Princeton, N.J.
[0114] Anticholelitholytic agents such as monoctanoin, Octanoic
acid, monoester with glycerol, Octanoic acid, monoester with
1,2,3-propanetriol, chemical registration no. 26402-26-6,
preparation and use described in U.S. Pat. No. 4,205,086 to
Babayan, May, 1980, marketed as Monoctanin.RTM. from Ethitek
Pharmaceuticals Co., Skokie, Ill.
[0115] The compounds of the invention can be assayed by any
techiques known in the art in order to demonstrate their
antiparasitic/antifungal activity. Such assays include those
described below in the Materials and Methods. Those compounds which
are demonstrated to have significant antiparasitic/antifungal
activity can be therapeutically valuable for the treatment or
prevention of leishmania, trichomoniasis, and trypanosomiasis.
[0116] Pharmaceutical compositions comprising the inhibitive
compounds or the salts thereof are provided by the present
invention. Administration of these compositions include, but are
not limited to, oral, intradermal, transdermal, topical, mucosal,
intravenous, subcutaneous, intramuscular, intraperitoneal, and
intranasal routes. More than one administration to the patient may
be necessary. The optimum amount of the antiparasitic/antifungal
agent varies with the weight of the patient being treated, with
some amount ranges presented in the patents describing these
agents. A range includes dosages of 0.1 mg/Kg/day to 50
mg/Kg/day.
[0117] A further embodiment of this invention includes the combined
therapy that can be obtained by treating patients with parasitic or
fungal diseases with a combination of the compounds of this
invention. The combination is preferably chosen such that the
inhibitory activity of the combined compositions is different, i.e.
the pathway is blocked at different points. The efficacy of
combined treatment could be substantially better than one
composition alone due to the ability to modulate different effects
of the compounds and possibly reducing side-effects or toxicity.
The administration of the compounds in the combination could be
simultaneous or sequential or in different dose forms including
combinations of oral dose forms with injectables to name just a
few.
[0118] The invention can be better understood by referring to the
following examples which are given for illustrative purposes only
and are not meant to limit the invention.
[0119] The following MATERIALS AND METHODS were used in the
examples that follow.
[0120] Trypanosome (a) IN VITRO drug screening method: 14 clinical
isolates of Trypanosoma brucei rhodesiense (agent of East African
Sleeping Sickness) many of which are refractory to standard
trypanocides such as diamidines and melarsoprol (Bacchi et al.,
1990, Antimicrob Agents Chemother 34, 1183-1188) are maintained as
laboratory infections in rats and mice and as frozen stabilates of
blood forms. Six of these isolates have been cultivated as blood
forms in modified Iscove's medium (Hirumi & Hirumi, 1989, J
Parasitol 75, 985-989). These cultivated blood forms are fully
infective and serve as the initial screen for trypanocides based on
a 24 well culture plate system and readings done on duplicate wells
using a Coulter Counter (Bacchi and Yartlett, 1993, Acta Tropica
54, 225-236; Bacchi et al., 1996, Antimicrob Agents Chemother 4,
1448-1453). This screen has proven highly reproducible, needs
minimal drug since the volume is limited (1 ml medium/well) and is
complete after 48 h. It has proven to be highly predictive of in
vivo activity (e.g., Brun et al., 1996, Antimicrob Agents Chemother
40, 1442-1447; Bacchi et al., 1996, supra).
[0121] Usually plant extracts are screened vs. four isolates, T. b.
brucei Lab 110 EATRO (veterinary parasite, and 3 strains of T. b.
rhodesiense (KETRI 243, KETRI 243 As10.sup.-3 and KETRI 269). The
latter are human clinical isolates refractory to arsenicals and
diamidines (KETRI 243 and 243 As10-3) or DFMO KETRI 269 (Bacchi et
al., 1990, supra). Extracts giving IC50 values at or below 100
ug/ml will be considered for further testing, using a more purified
extract.
[0122] Trypanosome IN VIVO drug screening method: Extracts having
significant activity in vitro (IC50 <10 ug/ml) along with
reasonable evidence of selectivity in mammalian toxicity tests will
be studied in in vivo screens. Initially the standard T. b. brucei
Lab 110 EATRO mouse model will be used. Agents proving active in
this model will be tested in the T. b. rhodesiense KETRI 243 and
269 model infections. These screens have proven effective in
identifying in vivo trypanocidal activities of DFMO and other
polyamine analogs, nuceloside analogs, arylguanylhydrzones and
other agents (e.g., Bacchi et al., 1980, Science 210, 332-334;
1987, Antimicrob Agents Chemother 31, 1406-1413; 1990, supra; 1992,
Antimicrob Agents Chemother 35, 2736-2740; 1997, Antimicrob Agents
Chemother 41, 2108-2112). Usually compounds are given by i.p.
injection once daily for 3 days. Animals surviving >30 days
beyond the deaths of untreated, infected controls with no evidence
of blood parasites are considered cured. Other routes used include
i.v., per os and Alza mini osmotic pumps which are implanted under
the skin and release 1 ?l of drug solution per h for 3 or 7 days.
Agents or extracts proving active in the above screens will be
studied in a standard CNS model infection (Jennings et al., 1983,
Contrib Microbiol Immunol 7, 147-154) which we have used to
demonstrate efficacy of single agents or drug combinations (e.g.,
Clarkson et al., 1983, Proc Natl Acad Sci 80, 5729-5733; Bacchi et
al., 1987, supra; 1996, supra), This model takes 10-12 months to
resolve and will only be used for highly active extracts and
purified substances.
[0123] TRICHOMONAD IN VITRO AND IN VIVO DRUG SCREENING METHODS.(a)
IN VITRO: Several strains of T. vaginalis are maintained, covering
the spectrum of metronidazole resistance: CDC85 (highly resistant);
RU384, RU383, IR78 (moderately resistant); NYH286, RU284, RU393,
C1-NIH (sensitive). These strains are routinely cultivated in a
non-defined medium incorporating tryptose, yeast extract, maltose
and supplemented with 10% horse serum.
[0124] In in vitro drug studies, plant extracts are tested using a
96-well plate assay as described by Meingassner et al., (1978,
Antimicrob Agents Chemother 13, 1-3). This method uses only 200 ul
of medium/well and thus uses very little plant extract. Results (in
triplicate) are presented as "Minimal Inhibitory Concentration"
(MIC) the lowest concentration completely blocking growth
(Meingassner et al., 1978, supra). This method is also useful in
comparing susceptibility of various strains (Meingassner et al.,
1978, supra; Yarlett et al., 1987, Mol Biochem Parasitol 24,
255-261). Assays will initially be done but highly active compounds
will also be tested anaerobically, since metronidazole resistance
is only detectable under aerobic assay conditions (Meingassner et
al., 1978, supra). Resistance to metronidazole is proposed to be
due to the presence of defective oxygen scavenging mechanisms and
resulting redox cycling of the partly reduced drug (Yarlett et al.,
1986, Mol Biochem Parasitol 19, 111-116). Since vaginal O.sub.2
tensions are {fraction (1/20)}th to 1/4th of atmospheric (Wagner et
al., 1978, Fertil Steril 30, 50-53), it is more physiological to do
drug sensitivity testing under conditions approaching this.
[0125] (b) IN VIVO: Extracts or highly purified material proving
active in vitro (MIC 0.5 mg/ml) with favorable selectivity will
also be tested in vivo in a mouse subcutaneous infection model
which has been used to correlate virulence of T. vaginalis isolates
with severity of pathogenicity in the human host (Honigberg et al.,
1966, Acta Cytol 10, 353-361; Kulda et al., 1970, Am J Obstet
Gynecol 108, 908-918). This model has been used successfully to
test various agents for trichomonacidal activity and is considered
superior to other in vivo tests (Brenner et al., 1987; Kulda, 1989,
Trichomonads Parasitic in Humans. Springer-Verlag, New York, pp
112-154).
EXAMPLE 1
[0126] Nineteen plant extracts were examined for activity in vitro
against four strains of animal or human-pathogenic African
trypanosomes, and three strains of mammalian-pathogenic Trichomonas
spp.
[0127] The trypanosomes studied were Trypanosoma brucei brucei Lab
110 EATRO, which is pathogenic to cattle and other livestock, and
several strains of Trypanosoma brucei rhodesiense, a parasite of
humans, domestic and wild animals. Strains of T. b. rhodesiense
included drug resistant clinical isolates KETRI 243 and 269 and
KETRI 243 As-10-3, a highly melarsen- and diamidine-resistant clone
of KETRI 243. The 19 extracts were tested in an in vitro screen
using a semi-defined medium for growth of bloodstream
trypomastigotes at 37.degree. C. (Hirumi & Hirumi, 1989, supra)
to determine IC.sub.50 values (Bacchi et al., 1996, supra). Using a
cutoff of 100 ug/ml, 12 of the 19 extracts consistently gave
IC.sub.50 values in the active range (Table 1). Of these, 10 had
IC.sub.50 values at or below 10 ug/ml and were considered
sufficiently active to warrant testing of more purified
extracts.
1TABLE 1 Activity of plant extracts vs growth of African
trypanosomes in vitro. Bloodform trypanosomes were grown in 24 well
culture dishes (1 ml/well) in HMI-18 medium (Hirumi & Hirumi,
1989, supra). One half of the culture volume was replaced daily
with fresh medium plus drug. Each extract was dissolved in 100%
DMSO and diluted with medium. Cells were counted daily with a
coulter counter. Data are as IC.sub.50 values in ug extract/ml
culture. Four strains were used: T.b. brucei Lab 110 EATRO, and
three T.b. rhodesiense clinical isolates from the Kenya
Trypanosomiasis Research Institute (KETRI). All data from 48 hr
cultures. Control cell counts averaged 5 .times. 10.sup.6 cell/ml
at 48 h. IC.sub.50 (ug/ml) KETRI 243 EATRO 110 KETRI 243 KETRI 269
As-103 SU-367 9.2 15.1 8.4 8.5 SU-369 11 5.1 8.2 11 SU-370 64 5 500
ug/ 500 ug/ ml-22% ml-22% SU-766 102 21.5 500 ug/ 47 ml-22% SU-787
9.0 8.5 12.5 14.9 SU-813 500 ug/ 500 ug/ 500 ug/ 500 ug/ ml-38%
ml-14% ml-44% ml-22% SU-614 134 74 79 51 SU-105 500 ug/ 500 ug/ 500
ug/ 500 ug/ ml-16% ml-8% ml-7% ml-8% SU-719 1.9 2.0 1.6 3.4 SU-679
18.0 19.5 28.9 40.5 SU-799 115 229 114 117 SU-740 33 32.5 30.0 39.0
SU-175 6.5 5.4 6.8 6.2 SU-847 13.5 8.3 12.5 12.6 SU-848 14.1 16.0
18.0 15.1 SU-769 119 73.0 74 78 SU-724 6.4 64.0 59 105 Pentamidine
0.00048 0.00186 0.00192 0.003 Melarsen 0.00077 0.0025 0.0066 0.0072
Oxide
[0128] Several secondary extracts were recently shipped and tested
(Table 2), while others are being prepared. Of the four secondary
extracts supplied, one, SU1460, derived from the primary extract
SU787 of Aframomum aulocacarpus, featured a 10-15 fold increase in
activity. SU787 had 1C50 values of 8.5-14.9 ug/ml (Table 1), while
the value for SU1460 was 0.86 ug/ml.
2TABLE 2 Activity of Secondary Plant Extracts on African
Trypanosomes. Assay method as in Table 1. Results as IC.sub.50 in
ug/ml. Primary Secondary IC.sub.50 Extract Origin Extract
T.b.brucei 110 SU-724 Araliopsis tabouensis AT6 SU-1459 500* SU-724
Araliopsis tabouensis AT7 SU-1458 100* SU-787 Aframomum
aulocacarpus AZ.sub.2 SU-1460 0.86 SU-175 Dracaena mannii SU-1461
6.4 Mannispirostan A *extracts precipitated in medium after 24
h.
[0129] Eight additional primary extracts were also tested in the
trypanosome screen (Table 3). Of these, SU1462 from Napoleonaea
imperialis and SU1464 from Glossocalyx brevipes were highly active
(IC.sub.50.about.1 ug/ml) and warrant further study.
3TABLE 3 Growth inhibitory activity of new primary plant extracts
against african Trypanosomes. Assay method as in Table 1. Results
as IC.sub.50 in ug/ml. IC.sub.50 Extract Origin T.b. brucei 110 SU
1462 Napoleonaea imperialis MeOH 1.75 SU 1463 Pachypodanthium
staudtii CH.sub.2Cl.sub.2 88 SU 1464 Glossocalyx brevipes
CH.sub.2Cl.sub.2 0.77 SU 1465 Enantia chlorantha MeOH 10.5 SU 1465
Eupatorium odoratum MeOH EOO 30% @ 50 ug/ml* SU 1467 Cleistopholis
patens EtOH 62 SU 1468 Leidobotrys staudii CH.sub.2Cl.sub.2 SU 1469
Ancistrocladus bateri ABSBM 28 *extract precipitates in medium at
higher concentrations
[0130] The trichomonad screen consists of two human pathogenic
Trichomonas vaginalis strains and a livestock parasite
Tritrichomonas foetus. The T. vaginalis isolates include a
metronidazole sensitive isolate (C1-NIH: ATCC 30001) and a strain
highly resistant to metronidazole (CDC-085: ATCC 50143). The
sceening procedure used is that of Meingassner et al. (1978, supra)
and determines the minimal inhibitory concentration (MIC) in mg/ml
needed to completely inhibit growth. Table 4 detail data from the
initial group of 19 primary extracts. Of these, seven had MIC
values of 1 mg/ml for all three isolates and were considered of
interest for further study. The results to Dec. 31, 1997 appear in
Table 5. The most active extract in this group was SU1464 from
Glossocalyx brevipes which had an MIC value of 0.0125 mg/ml for
each isolate and was the most potent of the primary extracts tested
thus far.
4TABLE 4 Minimum inhibitory concentration (mg/ml) of plant extracts
against Trichomonas vaginalis strain C1-NIH (ATCC#30001)
susceptible to current drug therapy (metronidazole and CDC-085
(ATCC#50143) resistant to metronidazole therapy; and the cattle
parasite Tritrichomonas foetus KV-1. Assays were performed in 200
ul multiwell plates (96 well) by serial dilution of each compound
(2.5 to 0.0012 mg/ml final concentration) and inoculated with 6.6
.times. 104 cells. Plates were scored after 48 h according to
motility (4 = 100%; 0 = no motility) compared to control wells
lacking the test compound (Meingassner et al., .1978, supra). MIC
(mg/ml) C1-HIH CDC-085 KV-1 ICBG# 48 hrs 48 hrs 48 hrs SU-105
>2.50 2.50 >2.50 SU-175 2.50 2.50 2.50 SU-367 12.50 12.50
0.78 SU-369 0.62 1.25 1.25 SU-370 2.50 2.50 2.50 SU-614 1.25 0.62
1.25 SU-679 0.62 0.62 0.62 SU-719 0.31 0.01 0.15 SU-724 0.62 0.62
2.50 SU-740 1.25 1.25 1.25 SU-766 1.25 1.25 2.50 SU-769 0.31 0.62
0.62 SU-787 0.62 1.25 2.50 SU-798 1.25 0.62 1.25 SU-799 0.15 0.31
0.62 SU-813 >2.50 >2.50 0.15 SU-846 2.50 1.25 2.50 SU-847
>2.50 >2.50 >2.50 SU-848 2.50 2.50 2.50 Metronidazole
0.003 0.40 0.004
[0131]
5TABLE 5 Inhibition of Trichomonas growth by new plant extracts.
Assay method as in Table 4. Data expressed as MIC in mg/ml. ND, not
determined. MIC Extract Origin C1-NIH CDC-085 KV1 SU 1463
Pachypodanthium 0.80 ND >0.80 staudtii CH.sub.2Cl.sub.2 SU 1464
Glossocalyx 0.0125 0.0125 0.0125 brevipes CH.sub.2Cl.sub.2 SU 1465
Enantia 0.80 ND 0.40 chlorantha MeOH SU 1467 Cleistopholis >0.80
0.10 >0.80 patens EtOH SU 1468 Leidobotrys 0.40 ND >0.80
staudii CH.sub.2Cl.sub.2 SU 1469 Ancistrocladus 0.40 ND 0.40 bateri
ABSBM Metronidaxole 0.003 0.40 0.003
[0132] The most active primary plant extracts in each screen are
listed in Table 6. These were chosen on the basis of MIC levels
(<1 mg/ml) for trichomonad screens and IC.sub.50 values
(</=10 ug/ml) for trypanosomal screens. Although many of the
extracts were most active only against one group of organisms, six
primary extracts had significant activity against both groups.
These were SU369, 719, 724, 787, 1464 and 1465. Of these, SU719 and
1464- appeared to be most potent in both screens.
6TABLE 6 Most active: ICBG primary plant extracts. Trichomonas
Trypanosomes (MIC < 1 mg/ml) (IC.sub.50 .ltoreq. 10 ug/ml)
SU-369+ SU-175** SU-679* SU-367 SU-719*+ SU-369+ SU-724+ SU-719**+
SU-769* SU-724+ SU-787+ SU-787**+ SU-799* SU-798** SU-1464*+ SU-846
SU-1462 SU-1465+ SU-847 SU-1464**+ SU-1469 SU-848 SU-1465+
[0133] Although large-scale testing of plant extracts for activity
against protozoan parasites is largely lacking (Wright &
Phillipson, 1990, Phytotherapy Res 4, 127-139) recent evaluation of
African medicinal plants vs. T. b. rhodesiense has given some
encouraging results (Freiburghaus et al. 1996a, J Ethnopharmacol
55, 1-11; 1996b, Trop Med Int Health 1, 765-771; 1997, Acta Tropica
66, 79-83). In these studies crude extracts were considered to have
promising activity in an in vitro screen against blood forms if
IC.sub.50 values were at or below 10 ug/ml. In the above
trypanosome screen 13 of 27 primary medicinal plant extracts had
such activity while two (SU719 and 1464) had IC.sub.50 values at or
below 1 ug/ml. Further studies will need to examine the selectivity
of active extracts, i.e. the maximum tolerated concentrations by
mammalian cell lines vs. the IC.sub.50 or MIC values. If the
selectivity data is favorable, further purification of the active
principles and animal testing would be the logical next steps in
the exploration of these extracts.
EXAMPLE 2
[0134] Using the leishmanial in vitro radiorespirometric bioassay
the active compound was purified and its structure determined. A
related species, Aframomum meleguata, showed moderate activity
against Trypanosoma brucei in vitro IC.sub.50 9.0 ug/ml. However, a
third plant species, Aframomum aulocacarpus, showed activity within
the highly active drug range, IC.sub.50 0.86 ug/ml, a 10-11-fold
increase in activity. The structural modifications in active
antiparasitic with these botanical species changes are in
progress.
EXAMPLE 3
[0135] Numerous similarities in leishmanial and trypanosomal lipid
uptake and metabolism may explain common natural product drug
susceptibility. Inhibitor, of cholesterol synthesis, metabolism,
and/or excretion described above in the detailed description were
tested versus trypanosome isolates grown as bloodforms in HMI-18
medium containing 10% fetal bovine serum. Coulter counds were made
daily and IC50 values determined after 48 h. Results are shown in
Table 7.
7TABLE 7 Drug compounds tested vs trypanosome isolates. Lab
IC.sub.50 (ug/ml) 110 EATRO 243 269 243 As 10-3 General inhibitors
Atromid-S >100 >100 -- -- Lopoid >100 >100 -- -- Bile
Acid resorption inhibitors Cholestipol >100 >100 -- --
Questran >100 >100 -- -- HMG-CoA reductase inhibitors Baycol
13 7.7 -- 52 Nevacor 3.3 4.4 6.9 -- Pravachol >100 >100 -- --
Zocor 1.33 12.9 7.0 -- Lescol >100 >100 -- -- Hormone
agorlists/antagonists Tamoxifen 30 Citrate* Tamoxifen* 27 Squalene
oxidase inhibitors Lamisil 1.3 86 77 >100 Thyroid hormone
antagonists Choloxin >100 >100 -- -- *uM
EXAMPLE 4
[0136] Targeted Anti-lipid Antileishmanials for Specialized Testing
in Primates.
[0137] Two compounds selected as inhibitors of cholesterol
synthesis and/or metabolism, and/or excretion will be tested at 3
dose levels in monkeys for evaluation against both monkey cutaneous
and monkey visceral leishmaniasis. In 4 experiments we want to test
2-drug-combinations (4 combinations) as antileishmanials. The
combinations we propose are already given in combination (for
nonleishmanial indications) to humans. These drug combinations
studies are in progress:
[0138] Positive Control Drug (Glucantime-Treated) Animals: (IP
Administration)
[0139] dose 1-13 mg/kg/day (MKD)
[0140] dose 2-52 mg/kg/day
[0141] dose 3-104 mg/kg/day
[0142] Negative Control (Suspending Drug Vehicle-HEC Tween Minus
Drug): (IP Administration)
[0143] PO Administration:
[0144] Drug 1 dose 1 (*human dose MKD level)
[0145] Drug 1 dose 2 (10.times. human dose MKD level)
[0146] Drug 1 dose 3: (10OX human dose MKD level)
[0147] Drug 2 dose 1 (*human dose MKD level)
[0148] Drug 2 dose 2 (10.times. humand dose MKD level)
[0149] Drug 2 dose 3: (10OX human dose MKD level)
[0150] Drug 1 dose 1+drug 2 dose 1
[0151] Drug 1 dose 2+drug 2 dose 1
[0152] Drug 1 dose 3+drug 2 dose 1
[0153] Drug 1 dose 1+drug 2 dose 2
[0154] Drug 1 dose 2+drug 2 dose 2
[0155] Drug 1 dose 3+drug 2 dose 2
[0156] Drug 1 dose 1+drug 2 dose 3
[0157] Drug 1 dose 2+drug 2 dose 3
[0158] Drug 1 dose 3+drug 2 dose 3
[0159] Candidate drugs 1 & 2 Vehicle Control (corn oil)
[0160] *Dose will vary depending on the drug being tested.
[0161] Discussion
[0162] Cholesterol is a sterol regulating the membrane fluidity of
eukaryotic membranes (Stryer, 1988, Biochemistry. WH Freeman and
Company, New York). Cholesterol contains a bulky steroidal nucleus
with a hydroxyl group at one end and a flexible hydrocarbon tail at
the other end (FIGS. 12-29, Stryer, 1988, supra). Cholesterol
inserts into membrane lipid bilayers so that the hydrocarbon tail
is located in the nonpolar core with the hydroxyl group bound to a
carbonyl oxygen atom of a phospholipid polar head group oriented
toward the aqueous exterior or interior of the cell (model previous
page). The interaction forces between sterol molecules seem to be
little affected by the double bond in the ring system or
modifications in the side chain. Also, the change in orientation of
the hydroxyl group from 3-beta to 3-alpha does not significantly
alter the cross-sectional area of the sterol at the surface.
However, replacement of the hydroxyl group by an oxogroup, or
changes in the planar structure of the sterol nucleus, increase the
molecular area, and may lead to some degree of membrane
destabilization. This is why certain dimerized natural product
plant components have antiparasite properties. Cholesterol prevents
the crystallization of fatty acid chains by fitting between them.
Thus, high concentrations of cholesterol tend to abolish phase
transitions of lipid bilayers (Bloch, 1983, CRC Critical Reviews in
Biochemistry 14, 47-92). Cholesterol (and sterol)-mediated
stabilization from phase transitions of lipid bilayers is
undoubtedly critical to the survival of Kintoplastida parasites
which must undergo marked temperature transition from ambient
(within the insect vector) to mammalian body temperature
(37.degree. C. or greater, dependent on reservoir or human
mammalian host) during their life cycle. Dependent on Tm, melting
temperature, fatty acid acyl chains in bilayers can exist either a
more rigid or ordered state favoring trans C--C bonds; or, at
rising temperature, a more disordered or gauche C--C bond
conformation (a 120-degree rotation, clockwise, g+, or
counterclockwise, g-) increases. The transition temperature, Tm,
depends upon the length of the fatty acyl chains and amount of
unsaturation. Saturated fatty acids result in an elevated Tm (e.g.
Crisco shortening, a solid at room temperature) whereas, greater
unsaturation increases fluidity (e.g. vegetable oils, liquid at
room temperature) lowering Tm. Likewise, cholesterol prevents
rigidity (crystalization) by fitting in between fatty acids
increasing fluidity, so that at high membrane cholesterol
concentrations, phase transition of bilayers are largely abolished.
An opposite effect of cholesterol is to sterically block large
motions of fatty acyl chains, making membranes less fluid. Membrane
fluidity, i.e. cholesterol content therefore, and indeed sterol
content in general, has strigent biologic control for each cell
type/function (Thompson, 1992, The Regulation of Membrane LiDid
Metabolism, CRC Press, Ann Arbor).
[0163] Medicinal herbs are of considerable importance to the health
of individuals and communities worldwide. Even in industrialized
countries, an estimated 33% of the population use alternative
treatments including herbal remedies. Approximately 35,000 to
70,000 plant species have been used for medical purposes (Zhang,
1996, World Health 49th year(2):4-5). Given the extraordinary ratio
(approaching 50%) of "active to total screened" plants developed
from our ICBG ethnomedical and ethnobotanical "leads" for
antiparasitics, one must be impressed by the accuracy of the
traditional healers' information. The fact that in the United
States, two thirds of the drugs currently available on the market
are originally based on medicinal plants then becomes somewhat less
astounding (Micozzi, 1996, World Health 49th year (2):8-9). Most
current antimalarials and other trypanosomals have their chemical
origins in herbal extracts, thus, scientific history would lead one
to believe that our ICBG approach is scientifically justified. The
data presented in this disclosure support the that conclusion that
the herbal extracts which, in fact chemically resemble various
components of sterol biosynthesis and metabolism, act by inhibition
of this pathway. The marked antiparasite efficacy of the known
anticholesterol, antihyperlipidemics, cholesterol hormone
antagonists, and anticancer drugs affecting this pathway for 3 of
the four human parasite genera we have studied to date, not only
provides immediate new chemotherapy for these infections in man and
animals, but supports the concept that efficacious and nontoxic
therapy for these diseases will be based on compounds affecting
this pathway.
* * * * *