Transcript Document

B-Specialized screening bioassays:
As described above, after having found a certain type of
activity, it will be necessary to study this activity in more detail by
using one or several specialized bioassays for screening and/or
monitoring purposes.
These in vitro or in vivo tests are more sophisticated than the
primary screening bioassays and require the expertise of biochemists
or pharmacologists. They can, consequently, only be performed in a
multidisciplinary team.
We have classified the many existing specialized screening
bioassays according to the target organisms which are used.
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1- Lower organisms:
Numerous research programs to detect and isolate
antibacterial-,
antifungal-,
antiviral-
and
antiparasitic
compounds from plants and other natural sources have been
designed and are performed.
Large batteries of bacteria, yeasts, fungi, viruses,
insects, molluscs, protozoa, and helminthes, are thereby used in
a broad range of in vitro and/or in vivo bioassays.
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Different microorganisms
Extract
dilution
control C
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2
C
3
C
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C
5
C
6
C
C
7 8
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10
C
C
C
11 12
C
C
A
1/2
B
1/8
C
1/32
D
control C
C
C
C
C
C
C
C
C
C
C
C
E
1/2
F
1/8
G
1/32
H
Scheme of a plate used in the agar dilution antimicrobial test method
Dr. Abd El Raheim Donia
2- Isolated subcellular systems:
Molecular assays look for activity using isolated systems, including enzymes and receptors.
Though already known for many years in pharmacology and frequently used in industrial drug
development, in studies of natural products the systematical use of these methods for screening purposes
has only started.
3- Isolated cellular systems:
Cellular assays utilizing intact cells of human or animal origin have been used for more than
half a century to screen for cytotoxic agents from natural sources. Cytotoxicity, or toxicity to cells in
culture, can be subdivided into cytostatic activity.
A large number of cytotoxicity- based bioassays have been used as prescreens for antitumor and
antineoplastic activity.
These short-term in vitro growth inhibition assays with cultured cells include Walker
carcinosarcoma 256, mouse L-120 leukemia.
While these traditional cell-cytotoxicity assays are indicative for activity in leukemia,
lymphoma, or a few rare tumors, their efficacy in finding products with activity in the predominantly
occurring slow growing solid tumors of humans is strictly limited.
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4- Isolated organs of vertebrates:
Organ-based assays are more and more replaced nowadays as
the front-line primary screen, but often retain signification as secondary
screens to confirm the results of, e.g. radioligand-binding bioassays and,
hence, to assist in prioritization of active extracts or compounds.
They represent, consequently, an essential connection between
the high technology of the primary screens and the realities of the
pharmacological effectiveness segments of the gastro-intestinal tract or
spirally cut strips of vascular tissue are mainly used in the organ bath
method for isolated organs.
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5- Whole animals:
Biomedical research needs animals. This is most obvious in the case of in
vivo animal experiments. However, for other scientific reasons, e.g. in vitro studies,
biological material is also needed to study enzymes, membranes, receptors, cells,
tissues or organs which are obtained from dead animals.
Therefore, animals have to be sacrificed in biomedical laboratories, (i) at
the end of in vivo experiments; (ii) during experiments where sacrifice of the animals
is not part of the study but must be done when pain, distress and suffering exceed
acceptable levels or if it is likely for the animal to remain in pain or distress after
cessation of the experiment; and (iii) to provide biological material for in vitro
studies.
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•
In vivo testing.
In vivo (Latin for "within the living") is experimentation using
a whole, living organism as opposed to a partial or dead organism,
or an in vitro controlled environment.
Animal testing and clinical trials are two forms of in vivo
research. In vivo testing is often employed over in vitro because it
is better suited for observing the overall effects of an experiment
on a living subject.
In vivo tests on animals often involve inducing a clinical
condition in the animal to produce observable symptoms.
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In vivo tests

There are several problems associated with in vivo testing.
*It is slow and it also causes animal suffering. There are
also many problems of pharmacokinetics and the result
obtained may be misleading.
For example, penicillin methyl ester is hydrolyzed in
mice into active penicillin, while it is not hydrolyzed in humans
or rabbits. Also, thalidomide is teratogenic in rabbits and
humans while it is not in mice.
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In microbiology in vivo is often used to refer to experimentation done in
live isolated cells rather than in a whole organism, for example, cultured cells
derived from biopsies. In this situation, the more specific term is ex vivo. Once
cells are disrupted and individual parts are tested or analyzed, this is known as in
vitro.
Compounds that bind to isolated recombinant proteins are one thing;
chemical tools that can perturb cell function another; and pharmacological agents
that can be tolerated by a live organism and perturb its systems are yet another.
If it were simple to ascertain the properties required to develop a lead
discovered in vitro to one that is active in vivo, drug discovery would be as reliable
as drug manufacturing.
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Most in-vitro screening assays utilize a surrogate readout scheme
that relies on labeled molecules or some type of coupled reaction as a
readout system. The biological relevance of these assay systems to the
original endogenous mechanisms can be tested with Isothermal Titration
Calorimetry (ITC) to compare the results and give confidence that they
mimic each other appropriately.
This includes all forms of ligand and receptor complexes, as well
as enzyme-substrate reactions and kinetics. Both ITC and Differential
Scanning Calorimetry (DSC) can be used to evaluate the quality of
expressed proteins for the appropriate post-translational properties and
percent activity, as well as assess the impact of assay co-factors.
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