Assignment on Preclinical and clinical screening of anti cancer drugs

1. INTRODUCTION
• Cancer is a disease characterized by uncontrolled
proliferation abnormally transformed cells
• There are more than 100 types of cancer
• A multifactorial disease
• Induction of proto-oncogenes and inhibition of tumor
suppressor genes has been implicated in the
pathogenesis of cancer
• Anticancer drugs are developed from variety of
sources ranging from natural products to synthetic
molecules

2. Drugs widely used as cancer chemotherapeutic agent suffers
from drawbacks like –
 High toxicity(bone marrow suppression, alopecia, nausea,
risk of secondary cancers)
 High cost


Development of resistance
Less tumor cell selectivity
 Necessiates development of compounds with lesser
toxicity, tumor cell selectivity, novel targets and more cost
effective
 For this quick and novel methods are being identified that
can screen a large number of compounds
 In vitro and in vivo models are systematically applied for
screening of anticancer drugs

3. PRECLINICAL SCREENING
In Vitro methods In Vivo methods

4. In Vitro Methods
Advantages
• Reduce the usage of
animals.
• Less time consuming,
•
•
Cost effective &
Easy to manage
• A controlled environment
can be maintained
• Able to process a larger
number of compounds
quickly with minimum
quantity.
Disadvantages:
• Difficulty in
maintaining of
cultures
• Show false positiveresults
• Show negative results
for the compounds
which gets activated
after body
metabolism
• Impossible toascertain the
Pharmacokinetics

5. • The goal of screening assay is to test ability of a compound
to kill cells
• Should be able to discriminate between replicating and
nonreplicating cell
• Different assays take advantage of various properties of cell
as mentioned below
CELL PROPERTIES ASSAY
ENZYMATIC PROPERTY MTT ASSAY
PROTEIN CONTENT SRB(SULPHORHODAMINE B) ASSAY
DNA CONTENT/REPLICATION STATUS 3
H-THYMIDINE UPTAKE & FLUORESCENT
ASSAY
MEMBRANE INTEGRITY DYE EXCLUSION TEST
COLONY FORMING POTENTIAL CLONOGENIC ASSAY
CELL DIVISION CELL COUNTING ASSAY

6. Microculture Tetrazolium Test(MTT)
• A quantitative colorimetric assay
• Measures cellular growth, cell
survival and cell proliferation
• Yellow Dimethyl thiazol
diphenyltetrazolium bromide a
tetrazolium salt is reduced to purple
formazan by mitochondrial
dehydrogenase of living cells
• Intensity of formazan produced is
directly proportional to cell viability

7. Cells from particular
cell line at log phase
of growth
tryptanised, counted
in haemocytometer,
adjusted to
appropriate density
Inoculated in
different mutiwell
plates, treated with
various conc of drugs
for specified
duration
MTT dye is added
and incubated at
37°C for 4 H in a
CO2 incubator
The percent of cell viability
with respect to control is
calculated using the formula
%cell viability=
(OD of treated cell/OD of
control cell)× 100
Plates are read
on an ELISA
reader at
570nm
Taken out of
incubator purple
coloured formazan
formed thoroughly
mixed with
isopropanol/DMSO

8. Sulphorhodamine B Assay
• Measures whole culture protein content
• SRB is a bright pink anionic protein staining dye that
binds to basic amino acid of cell.
• Cell cultures are stained with it and unbound dye is
removed by washing with acetic acid
• Determination of optical density in a computer
interfaced,96-well micro titer plate reader
• The amount of SRB binding is proportional to the
number of live cells left in a culture after drug
administration
• Screening capacity, reproducibility, quality control all
appear to be enhanced

9. 3
H-thymidine uptake assay:
• Replicating cells will incorporate H-thymidine which then can be
determined by autoradiography or liquid scintillation counting
3
• Provides information on tumor growth kinetics
• DNA Histogram-information on ploidy status of cell
Fluoroscence:
• Fluorescent dye incorporation followed by microcsopic evaluation
• Replicating cells incorporate labeled precursor into their DNA and
resulting Fluoresence is measured by flow cytometry
Dye Exclusion Test:
• Relied on structural integrity of cell.
• Dead cells have lost membrane integrity and would take up vital dyes
like tryptan blue
• Modified version-Differential staining cytotoxicity assay
• End point morphologic identification of tumor cell cytotoxicity
compared with internal control

10. Clonogenic assays:
• Measure tumor cell reproductive viability
• Most direct method of measuring cytotoxicity of a drug
Cell counting assay:
• Cells are cultured in the presence of drugs for 2-5 culture doubling
times
• Cell number is estimated using a haemocytometer or cell counter
3D Tumor Models:
• Cancer cells are cultured in a spatially relevant manner with
endothelial cells and other cells
• Biomimetic property accurately depict in vivo situation of drug
screening
• Advantageous over complexity of animal models and the spatial
limitaion of cell culture model
4D Tumor Model:
Ex vivo lung cancer model of perfusable nodule on a lung matrix

11. Examples of 3D models
Spheroid system:
• Three dimensional multicellular tumors derived from HeLa cell
• Quantify chemotherapeutic and nanoparticle penetration in
vitro
• Acquire several clinically relevant morphologic and cellular
characteristic often found in human solid tumors
Spherochip system:
•
•
•
•
•
Automated assay
Microfluidic based platform for long term 3D cell culture
Analysis is compatible with commercially available microplate
readers
Dynamic change in metabolic activity of cell can be observed

12. In Vivo methods
• Aimed at predicting
Safe starting dose & dosage regimen for human clinical trials
The toxicities of the compound, &
The likely severity and reversibility of drug toxicities
Advantages:
• Detect host mediated
activity
• Relatively predictable
• Estimate therapeutic
ratio
• Used for both
preclinical anticancer
efficacy detection and
for
toxicological studies
Disadvantages:
• Sensitivity is low
•
•
•
Costly
Time consuming
Large number of
samples cannot be
handled
• Difficult to manage

13. Chemically Induced tumor
models:
• DMBA-induced mouse skin
papillomas, rat mammary
gland carcinogenesis, oral
cancer in hamster
• MNU-induced rat
mammary gland
carcinogenesis, tracheal sq
cell CA in hamsters,
prostate cancer in gerbils
• DEN-induced lung
adenoCA in hamster
• DMH-induced colorectal
adenoCA in rat and mouse
• OH-BBN induced bladder
CA in mouse
• Hepatocelluler CA models
Models involving cell
line/tumor pieces
implantation:
• Cell line implantation
•
•
•
•
•
Hollow fiber technique
Use of Xenografts
Nude mouse model
Newborn rat model
Transgenic mouse
model
Viral infection models:
• Mouse mammary
tumor virus
• Moloney murine
sarcoma virus
• Newer genetically
engineered viruses

14. DMBA-induced Mouse skin papilloma
• classical two stage experimental
carcinogenesis model
• SENCAR mouse is highly sensitive-tumor
incidence 100% in controls
• Dimethylbenzanthracene(DMBA) acts as
initiator and tetradecanoyl phorbol
acetate is used as promoter
• Topically applied on shaved back till
appearance of papillomas (6-7) weeks
• Weekly monitoring of tumor development
for 18 weeks
• Percent tumor incidence and multiplicity
are compared between treatment and
DMBA control groups.
Mouse skin papilloma

15. MNU induced rat mammary gland CA
• Induces hormone dependent
tumors.
• Single i.v of 50 mg/kg of
methylnitrosourea(MNU) given to
50 days old Sprague-Dawley rats.
• Adenocarcinoma will be produced
within 180 days of post carcinogen
in 75 to 95% cases
• Drug efficacy is measured
• Drawback – cannot detect inhibition
of carcinogen activation

16. Other similar models
Cancer site Cancer Type Species Carcinogen
Colon Adenocarcinomas Rat, Mouse AOM (azoxymethane),
DMH
(Dimethylhydralazine)
Prostate Adenocarcinomas Gerbil MNU (methylnitrosourea)
Trachea Squamous cell
carcinoma
Hamster MNU (methylnitrosourea)
Breast Adenocarcinoma Mouse NMU (methylnitrosourea)
DMBA
(Dimethylbenzanthracene)
Lung Adenocarcinoma Hamster DEN (Diethylnitrosoamine)

17. Hepatocellular carcinoma models
• Can be readily induced by chemical carcinogens
• Several animal models are well established
• Naturally occuring- Wood chuck, Long Evans Cinnamon
rats
• Ethylnitrourea induced HCC in B6C3F1 mice is widely
used due to
easy maintenance
consistency of results
long duration of study is comparable to human situation
• MDR2 Knockout mice: lack Pgp in bile cannaliculi
develops hepatocellular carcinoma.

18. Methods involving cell line:
• Specified number of particular cell line inoculated into sensitive mouse
strain
• Tumors develop rapidly thus time saving
• Effective drug retard tumor growth and increase life span of animal
• L-1210, P-388, B-16 cell lines- host mouse strain BDF1
• Sarcoma-180 – Swiss albino mouse
Hollow Fiber Technique:
• Small hollow fibers containing cells
from human tumors
• Inserted underneath skin and in body
cavity of mouse
• Candidate drug tested in vivo
• Compounds retarding growth
are recommended for next level of testing
Subcutaneous hollow fiber implant

19. Xenografts
• Human tumors(lung, breast, colon, ovary, brain, HCC) are
optimized in mouse cell lines
• Directly injected below the skin of the mouse
• Drugs showing activity in hollow fiber model are administered at
various dosages
• Compounds that kill or slowdown growth of specific tumor with
minimal toxicity-procced to next stage of testing
• 1. Spheroid culture of LuCap 147-induced prostate cancer model
2.Integration free-induced pluripotent stem cell
model
high throughput screening
drug induced cell cycle arrest
apoptosis
can be demonstrated in spheroid cultures

20. Nude mouse:
• Immunologically incompetent mouse due to absence of thymus
• Do not show contact sensitivity
or reject the transplant material
• Melanomas, colon carcinomas
grow very well
Newborn rat model:
• Can be used as an alternative to nude mouse
• as cost effective and maintenance is easy
Transgenic mouse model:
• Inactivation of a particular gene within specific tissues of adult mouse
• Serve as both model of disease as well as gene therapy
• Metamouse: tumor pieces of patients are directly transplanted to
organ of primary growth
• Metastasis and weight loss occurs same way as in humans
• Test new routes, doses and indications of old drugs

21. Summary
• Focus has shifted from cytotoxic compounds to target
based therapy
• Basic research in cancer biology has provided new targets
for drug development and brought older targets to
sharper focus
• Newer treatment regimen, routes of administration,
approaches are increasingly evaluated
• Current challenges are significant time and cost
involvement and low success rate
• For this faster and more predictable screening methods
are being developed
• Continuous improvement now is incorporating a risk
based approach

22. Thank You