PHARMACOGNOSY II

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PHARMACOGNOSY II for DOCTOR of PHARMACY (152425)
by Nisit Pisutthanan
room Chaiya 4 date 20/8/2004
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Tumour Inhbitors from Natural Products
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TUMOUR ?A mass of excess tissue that results from
abnormal cell division. Tumors perform no
useful body function. They may be benign (not
cancerous) or malignant (cancerous).
CANCER ?A term for diseases in which abnormal
cells divide without control. ?Cancer cells
can invade nearby tissues and can spread
through the bloodstream and lymphatic system
to other parts of the body.
4
Main types of cancer Carcinoma is cancer that
begins in the skin or in tissues that line or
cover internal organs. Sarcoma is cancer that
begins in bone, cartilage, fat, muscle, blood
vessels, or other connective or supportive
tissue. Leukemia is cancer that starts in
blood-forming tissue such as the bone marrow, and
causes large numbers of abnormal blood cells to
be produced and enter the bloodstream. Lymphoma
is cancer that begins in the cells of the immune
system.
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Introduction
?The main forms of treatment for cancer
surgery, radiation, chemotherapy and
immunotherapy. . ?Many hundreds of chemical
variants of known classes of cancer
chemotherapeutic agents have been synthesized
for more activity and selectivity. . ?New
prototypes or new templates are needed to use in
the design of potential chemotherapeutic
agents natural products are providing such
templates. gtgtgtgt novel structures and extremely
complex ltltltlt
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Role of Plants in Cancer Treatment
?Plant materials have been used in the treatment
of malignant diseases for centuries. (in
folklore) ?Phytochemical examination of such
plants has often resulted in the isolation of
principles with antitumour
activity. ?Podophyllum ancient Chinese
antitumour drug dried rhizome and roots of
Podophyllum peltatum (Berberidaceae) yield
Podophyllum resin about 2-8.
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The rhizome, which is about a metre in length,
is dug up, cut into pieces about 10 cm in
length, and dried.
Uses Podophyllin (a crude resin) vermifuge
and emetic purgative treatment of venereal
warts antitumour properties developed to be
anticancer agents
Podophyllum peltatum (May-apple, Wild Mandrake)
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Chemical constituents
?-Peltatin, R CH3 ?-Peltatin, R H
Podophyllotoxin topically to treat venereal warts
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Etoposide
Lignan derivative obtained semi-synthetically
from podophyllotoxin. Used in the treatment of
small-cell lung cancer,
testicular cancer,
lymphomas and leukaemias.
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Teniposide
Thenylidene derivative Used in the treatment
of paediatric cancer
neuroblastoma,
lymphocytic leukaemia,
brain
tumours.
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Traditional medicines for treatment of cancer
Solanum dulcamara (woody night-shade) the
expressed juice of this plant has been used to
treat cancers, tumours and warts in many
countries. active tumour-inhibitory
principle gtgt b-Solamarine
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b-Solamarine steroidal alkaloid glycoside
aglycone tomadidenol sugar chacotriose
(glu.-rhamn.-rhamn.)
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Lichens (Cladonia, Cetraria, Usnea) have a
history of use in folk medicine against
cancer. Usnic acid, has recognized for many
years as an antibacterial and antifungal
agent, but more recently as an antitumour
compound. Usnic acid has been shown to
exhibit antiviral, antiprotozoal,
antiproliferative, anti-inflammatory,
analgesic and antipyretic activity.
Ecological effects, such as antigrowth,
antiherbivore and anti-insect properties,
have also been demonstrated.
(Phytochemistry. 2002 Dec61(7)729-36, Planta
Med. 1995 Apr61(2)113-5)
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Usnea barbata (Usneaceae)
Lichen symbiotic combination of alga and fungus
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Usnea diffracta (Usneaceae)
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Usnic acid (usnein, usniacin)
It is used as an antibacteria agnet in cosmetics
and ointments to prevent skin infection.
It is also used in formulating deodorant,
mouth-care products.
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Viscum album (Viscaceae) Mistletoe
protein fractions with marked antitumour
activity have been isolated from mistletoe
extract. phase II study of mistletoe ext. in
patients with advanced hepatocellular
carcinoma. (Br J Cancer. 2004 Jan
1290(1)65-9)
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Structure of Mistletoe Lectin I from Viscum
album (if you want to see, please click here)
The structure was accessed from
http//www.ncbi.nlm.nih.gov/ Structure/mmdb/mmdbsr
v.cgi?form6dbtDoptsuid26400
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Daphne mezereum (Thymelaeaceae) Mezereon
all parts are highly toxic but has been used
un many countries for the treatment of
cancer active cpd. diterpene derivative of
mezerein
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Mezerein antileukaemic, tumour promotor
(protein kinase C activator)
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Catharanthus roseus (Apocynaceae) Madagascan
periwinkle
folk medicine treatment of diabetes showed
leukopenic actions in rats intensive
investigation by Eli Lilly obtained six
alkaloids with anticancer property more than
150 alkaloids have been isolated (20 bisindole
alkaloids)
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Chemical constituents
Vinblastine, R CH3 Vincristine, R CHO
Vinblastine Hodgkins disease and
non-Hodgkins lymphomas Vincristine
acute lymphatic leukaemia in children
and other combination regimens (small
cell lung cancer, cervical and breast cancer)
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How to increase the amount of vincristine?
A
B
vincristine
vinblastine

• Convert by chemical reaction
• B . Convert via microbiological transformation
• Streptomyces albogriseolus

Cell cultures total alkaloids 0.1-1.5 dried
wt. catharanthine and tabersonine, not vindoline
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Vindesine semi-synthetic derivative
acute lymphoid leukaemia in children
vinblastine
vindesine desacetyl vinblastine amide
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Potential Toxicity
Preparation Vinblastine
iv Vincristine
iv Vinorelbine
oral
vinblastine
vinorelbine
Toxicity neurotoxic side effects
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Methods of investigation
?In the late 1950s, an intensive survey of
plants, microorganisms and marine animals for
antitumour agents was began by US NCI. ?Using a
random-selection screening programme. ?114,000
plant samples from 40,000 species. ?About 4 of
the extracts have shown reproducible. ?About half
a million plant species exist worldwide, thus
the plant kingdom still represents a vast
untapped source of material.
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The term cytotoxic / antitumour / anticancer?
A cytotoxic agent is toxic to tumour cells in
vitro and if this toxicity transfers through to
tumour cells in vivo, the agent is said to have
antitumour activity. The term anticancer is
reserved for materials which are toxic to tumour
cells in clinical trials.
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The isolation of biologically active
constituents ?Conventional phytochemical
evaluation -to study cpd. which were most
easily separated (present in the largest
quantities and crystallized readily) -to study
the specific group of interest (e.g.
alkaloids, terpenoids, phenols, etc.) -to
elucidate the isolated compounds -to test for
their biological activities (this would
depend on sufficient material being
available) gtgtgtCountless medicinally useful cpd.
have been missed in this type of approach.ltltlt
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The isolation of biologically active
constituents ?Systematic investigation -every
portion of the plant and every fraction of
the extract is biological tested before isolation
or characterization -any class of active
cpd. may be isolated -may not be traditionally
associated with a particular plant family
gtgtgtgtgtgtbioassay-directed
fractionationltltltltltltlt
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No problem in this type of approach?
?Isolated constituents of a plant drug may not
give the same clinical response as a crude
preparation of that plant drug. ?Synergism or
antagonism due to the complex nature of the
extract are probably the causes of such
observation. ?A further complication is that
crude fractions may contain additional
substances with delayed toxicity, causing test
animals to die at about the same time as
control animals.
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Methods of investigation
An effective screening procedure should fulfil
several criteria including selective --to
limit the number of lead for
follow-up evaluation sensitive --in order to
detect low conc. of active
cpd. specific --so that the assay is not
affected by a wide variety
of inactive cpd.
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The preliminary screening programme of NCI
gtavoid weakly active common metabolites
tannins, saponins, sterols gtthe routine testing
(for initial tests) in vitro cytotoxicity
rapid, inexpensive, need small amount of
extract KB cytotoxicity assay, human
epidermoid carcinoma of the nasopharynx
(milligram of sample was need, whereas in
vivo may require 0.5-1 gram) gtcurrent preliminary
screens in vivo mouse P-388 lymphocytic
leukaemia system in vitro KB assay gtextracts
showing activity in these systems are candidates
for fractionation studies. (may use in vitro
P-388)
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The fractionation procedure to obtained active
cpd.
Bioassay-directed fractionation
Solvent partitions, Weak acids and bases
partition Chromatography (column and planar)
problems?
Structure elucidation of active cpd. using MS,
NMR, IR, UV and X-ray crystallography
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What should be done with the active cpd.?
tested against a range of standard experimental
neoplasms
preclinical toxicological studies (large amounts
of material will be required)
(high therapeutic index with low side effects)
clinical studies
35

number of screens Preliminary screening test
25,000 (per year) Preclinical testing
8-12 Clinical
trials 6-8
Slightly less than half of these may be
plant-derived.
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Availability of supplies of plant materials?
Should a plant-derived natural product or
derivative be considered worthy of development as
a drug!
Alternative plants
Plant tissue culture
Total synthesis
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The random-selection screening programme
for natural products was terminated by the NCI
in 1983. the programme had not identified a
single agent for general use in the treatment
of human cancer. a large number of cytotoxic and
antitumour agents had been identified. increase
in the understanding of the cancer process and
the mechanisms of action of agents. synthetic or
semi-synthetic analogues and SAR have been
undertaken. Advanced clinical trials --
established the cpd. in the NCI programme to
provide new cancer drugs, including taxol and
camptothecin.
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With a confidence in the potential of natural
products as leads for new anticancer agents, a
new screening system was begun in
1986. reducing the scale of the
operation collecting plants of a board taxonomic
range from tropical to subtropical parts of the
world giving particular emphasis to the
collection of local medicinal plants.
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Project 1 (Plant selection, Acquisition
Inform. Mgmt.)
Project 2 (Extraction, Isolation Structue
Elucidation)
Project 5 (Synthesis)
Project 3 (In vitro bioassay)
Project 4 (In vivo bioassay)
Interrelationships of projects aimed toward the
discovery of natural product cancer
chemotherapeutic agents.
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Screening for cytotoxic metabolites Primary
screening bioassay - Brine shrimp lethality
tests - Crown-gall tumor bioassay - Starfish
or sea urchin assay Specialized screening
assays - Cytotoxicity - Antitumour -
Antineoplastic activity
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Established natural products as tumor inhibitors
Table 1 the tumour-inhibitory principles
isolated in screening tests were usually new
natural products, spanning a wide range of
structure types. (some were previously known
cpd. including usnic acid, lapachol,
retronecine, monocrotaline, hellebrigenin,
colchicine, cucurbitacins) Figure 1 the
structures of the more compromised chemicals
which were subsequently evaluated in clinical
tests.
42
According to the preliminary screening tests
in most cases, the activity levels noted in
the screens were not sufficient to justify
further evaluation lapachol entered clinical
trials but lack of therapeutic
response and blood
anticoagulant side-effect
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incidiene-N-oxide showed no evidence of
hepatotoxicity possessed significant
antitumour activity in clinical trial, showed
substantial activity in acute leukaemia, but
hepatotoxicity was more severe than expected.
Heliotropium indicum (Boraginaceae)
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cucurbitacins some of them showed highly
cytotoxic compounds this highly
cytotoxicity carries over to in vivo systems
narrow therapeutic index
Marah oreganus (Cucurbitaceae)
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colchicine widely used in plant breeding
(mitotic poison) a large number of natural
and semi-synthetic derivatives have been
tested, several in clinical trials, but the
results have been disappointing. demecolcine
is being used in cancer treatment in some
countries
Gloriosa superba
Colchicum speciosum
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podophyllotoxin deri., vinblastine, vincristine
also act as mitotic poisons. major
successes in drug control of cancers in the
area of rapidly-growing tumors. slow-growing
tumors generally seem to respond poorly to
drugs.
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Further examples of known cpd. as tumour
inhibitors ?Alkaloids acronycine, ellipticine ,
emetine, nitidine
acronycine acridone alkaloids was first
isolated in 1948 but its activity was
reported in 1966 exhibited a very broad
antitumour spectrum, possessing significant
activity against 12 of the 17 tumour systems
tested. in marked contrast, phase 1 clinical
trials displayed no significant antitumour
activity. gtgtgtthe obvious discrepancy is
attributed to the sensitivity of the
preliminary screens appliedltltlt
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Clinically useful antitumour agents were based on
the pyridocarbazole alkaloids ellipticine and
9-methoxyelleipticine
-potent inhibitors -broad spectrum -preclinical
side-effect haemolysis, cardiovascular
effect -mechanism of action DNA
intercalation, inhibition of topoisomerase II
and recently, formation of covalent DNA
adducts in vitro. (Int J Cancer. 2003 Dec
20107(6)885-90)
Ochrosia elliptica (Apocynaceae)
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water soluble N-glycosides
highly active against breast cancer and also
renal cell cancer
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Cephaelis ipecacuanha has been used as an emetic
and expectorant treatment of amoebic dysentery
emetine isoquinoline alokaloids it and
its semi-synthetic derivatives were tested
clinically clinical usefulness was
marginal, however, some toxic effects were
noted.
Cephaelis ipecacuanha (Rubiaceae)
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nitidine (Fagara macrophylla, Zanthoxylum
nitidum) benzophenanthridine alkaloid was
selected for development based on its
exceptional anti-leukaemia activity was
dropped owing to erratic (uncertain)
toxicity fagaronine less toxic than
nitidine Fagara zanthoxyloides
Zanthoxylum nitidum (Rutaceae)
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More recently, NK 109, a synthetic isomer of
fagaridine found in Fagara xanthoxyloides, has
been found to have greater antitumour activity
than any of the natural benzophenanthridine
structures, coupled with excellent stability,
and has entered clinical trials in Japan.
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New natural products with antitumour activity
The systematic studies have also resulted in the
isolation of many new natural products
exhibiting antitumour activity. A number of
them have been considered sufficiently active
for clinical studies to be commenced. (table 1
and figure 1)
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Tylocrebine phenanthroindolizidine
alkaloid Tylophora crebiflora (Apocynaceae)
was sufficiently active for further
development but in a clinical trial
unmanageable CNS effects precluded
(prevent) continuation of the studies.
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thalicarpine (Thalictrum dasycarpum,
Ranunculaceae) and tetrandrine (Cyclea peltata,
Menispermaceae) bis-benzylisoquinoline
alkaloids clinical trials showed no
antitumour actiivity
gtgtthese cpd. were isolated because the
screening were too sensitiveltlt
on the other hand, tetrandrine was found to
be able to block calcium channel.
Thalictrum dasycarpum
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camptothecin and derivatives
pyrroloquinoline alkaloids showed broad
spectrum activity produced a fair response in
limited clinical trials toxicity and
poor solubility 10-hydroxycamptothecin is used
in China against cancers of the neck and head
Camptotheca acuminata (Nyssaceae)
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Synthetic analogues
water soluble
ovarian cancer
carbamate pro-drug of 10-OH-7-ethylcamptothecin co
nverted into the active drug by liver enzymes
water soluble
colorectal cancer
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Quassinoids or simaroubolides a group of
terpenoid-related cpd. isolated from a
variety of plants in the Simaroubaceae many
species have been used for anti-amoebic in
folk medicine a number of quassinoids are
currently of interest for their antitumour
properties Brucea antidysenterica used to
treat cancer in Ethiopia systematic
fractionation has led to isolate bruceantin,
shows high antileukaemic activity at low dose
acts through inhibiton of protein
synthesis research on quassinoids related to
bruceantin continues
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Maytansinoids ansa macrolide Maytenus
serrata and other species of Maytenus mosses
and Actinomycete
Maytansine high yield, active against
several neoplasms at very low dosage (mg/kg
animal b.w.) over 50-100-fold dosage range
inhibition of mitosis in clinical trials
--big appointment synthetic and
semi-synthetic derivatives may offer more
hope
Maytenus ilicifolia (Celastraceae)
Recent developments in the maytansinoid antitumor
agents. Chem Pharm Bull (Tokyo). 2004
Jan52(1)1-26.
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Maytansinoids could be produced via
microorganisms A species of Nocardia has been
shown to produce ansamitocin , which is a
mixt. of esters of maytansinol (the parent
alcohol of maytansine) Ansamitocin could be
served as starting material for semi-synthesis
of a whole range of derivatives.
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Natural products with sufficiently interesting to
justify clinical trials or toxicological
testing. Triptolide and tripdiolide diterpene
potent antileukaemic agents contained
reactive triepoxide low percentage yield
Nevertheless, in China crude extract of this
plant is used in inflammatory and immune
disorders, and triptolide is currently
undergoing evaluation for these disorders.
Tripterygium wilfordii (Celastraceae)
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Sesquiterpene lactones many of them were tested
but few show useful in vivo antitumour activity
e.g. elephantopin (germanacrolide
sesquiterpene) from Elephantopus
elatus baccharin (trichothecene sesquiterpene)
from Brazilian Baccharis megapotamica related
to fungal trichothecene toxins baccharin and
some of the fungal-derived cpd. are
undergoing through evaluation
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Alkloidal esters from Cephalotaxus spp. are
currently being isolated on a large scale for
toxicological studies. harringtonine and
homoharringtonine show good activity in a
number of systems favorable results in
clinical studies using crude alkloidal
fractions of C. harringtonia and
homoharringtonine (in particular) in
patients with leukaemia resistant to
existing chemotherapies. homoharringtonine is
a minor constituent but can be obtained by
semi-syn. from the more abundant
cephalotaxine.
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Cephalotaxus harringtonia (Cephalotaxaceae)
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Phyllanthostatin 1 and phyllanthoside
(sesquiterpene) a complex mixt. with marked
antitumour properties obtained from the root
of central American tree, Phyllanthus
acuminatus phyllanthoside is in early
clinical trials.
Phyllanthus acuminatus (Euphorbiaceae)
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Combrestatin A-4 (cis-stilbene) one of the
most potent antimitotic agents from about 20
active substances isolated from African tree
Combretum caffrum (Combretaceae). a
water-soluble phosphate pro-drug is now in
clinical development. tubulin polymerization
inhibitor and interfering with the blood
supply of tumors.
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One of the most promising plant-derived
anticancer agents to be identified in recent
years is the taxane diterpenoid taxol
(paclitaxel) isolated from the bark of
Pacific yew tree, Taxus brevifolia
(Taxaceae). very low yield (0.01-0.03) the
bark from 3 trees is needed to produce 1 gram
of taxol!
Taxus brevifolia
68
Taxol could be obtained using semi-synthesis
from other taxane derivatives. 10-deacetylbaccati
n III, isolated from needles of T. baccata
higher yield 0.1-0.2 A side-chain analogue
of taxol, known as taxotere, has also been
developed by semi-syn. from
10-deacetylbaccatin III. improved water
solubility and better activity than taxol
in some assays. is currently used against
resistant breast cancer.
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The NCIs fractionation schemes based on a
balance between hydrophilicity and
lipophilicity for substances to reach
receptors. extremely polar and non-polar
extracts are unlikely to show as much activity
as extracts of intermediate polarity.
recently, many peptides and proteins from micro-
organisms, and some water-sol. polysaccharides
have shown high antitumour activity.
gtgtconsideration is thus being given to the
testing of aqueous extracts from plantsltlt
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Already, screening of the Mexican plant
Bouvardia ternifolia (Rubiaceae) yielded the
bicyclic hexapeptides, bouvardin and
deoxybouvardin as active principles. was
selected to further development was dropped
because of its narrow spectrum of activity
Bouvardia ternifolia
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Future developments
The search for new antitumour cpd. in nature is
by no means confined to plants. Microoganisms,
widely employed sources of antibiotic, produce
a considerable number of metabolites having
antitumour activity. actinomycin D,
bleomycin, adriamycin, daunorubicin,
mitramycin and mitomycin C. have particular
advantages ease of culture and the
opportunity for genetic manipulation. the
recent identification of epothilones from the
bacterium Sorangium cellulosum some analogues
are much more potent than taxol, especially
towards multi-resistant cell lines.
72
Marine animals may also be a fruitful source of
potentially useful anticancer agents.
didemnin B, a cyclic depsipeptide from
Caribbean sea-squirt, Trididemnum solidum
gtactive against prostate, lung, brain cancer,
lymphomaslt
Sea-squirt
Structure of sea-squirt
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bryostatins, a group of macrocyclic lactones
from marine bryozoan, Bugula neritina
gtcause tumor destruction at the cellular level by
transporting chelated cations across the cell
membranelt gtshowed enormous potential for inh.
protein kinase C tumor promotionlt
Bugula neritina
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dolastatin 10, a linear peptide from sea hare,
Dolabella auricularia
gtvery potent inhibitor of microtubule
assemblylt
Dolabella auricularia
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A natural product of high interest is
4-ipomeanol, a toxin isolated from
mould-infected sweet potato, Ipomoea batatas,
which, when ingested, affects the lungs and
kidneys of mammals. gtgtgtphytoalexinltltlt 4-Ipomean
ol is selectively oxidized by enzymes in
certain lung cells to give a reactive dialdehyde,
and this provided a powerful alkylating agent
only in those cells. 4-Ipomeanol is undergoing
clinical trials against lung cancers.
76
Targeting of cytotoxic agents or toxins to
cancer cells or tumours using monoclonal
antibodies is also receiving considerable
attention. Some success has been achieved in
animal studies using the castor seed toxin,
ricin bound to monoclonal antibodies.
77
The current NCI screens against 60 different
cell lines representing a variety of human
tumour types spanning --leukaemias, melanomas,
and tumors of the lung, colon, kidney,
ovary, breast, prostate and brain. developed
from knowledge of the basic biological
mechanism of action of known clinically
effective anticancer agents --tubulin binding
(vincristine, vinblastine) tubulin
stabilization (taxol) topoisomerase I
(camptothecin) topoisomerase II (etoposide)
78
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