Screening methods for Anticancer drugs. Description of Principles and procedures of screening methods for anticancer drugs.

1. Priyanka Sanas
Master of Pharmacy: Pharmacology
1st Year

2. 
 What is Cancer
 Tumors
 Types Of Cancer
 Need for Novel Anticancer Agents
 In Vitro Models
 In VIVO Models
Contents..

3. 
Cancer may regarded as a group of diseases
characterized by an Abnormal growth of cells which
possess Ability to invade tissue and even distant
organs.
What is Cancer…???

4. 
 An abnormal growth of tissue resulting from
uncontrolled, progressive proliferation generates a
mass of cells is called tumor.
 Two Types of tumors are seen:
1. BENIGN Tumor
2. MALIGNANT Tumor
Tumors

5. 
 Carcinoma: Cancer of Epithelial Tissue.
 Sarcoma: Cancer of connective tissues, like bones,
cartilages, blood vessels or muscles.
 Leukemia: Cancers that start in blood forming
tissues such as bone marrow.
 Lymphoma & Myeloma: Cancer in cells of the
Immune system.
 Glioma & Astrocytoma: Cancers of the central
nervous system that arise in the tissues of the brain
and spinal cord.
Types Of Cancer

6. 
 Development of multidrug resistance in patients.
 Long-term treatment with cancer drugs is also
associated with severe side effects.
 Cytotoxic drugs have the potential to be very
harmful to the body unless they are very specific to
cancer cells.
 New drugs that will be more selective for cancer
cells.
Need for Novel
Anticancer Agents

7. 
 Brine Shrimp Lethality Bioassay of compounds
 Trypan blue exclusion assay on murine cell lines
 Cytotoxicity assays on panel of human cancer cell
lines: MTT-assay AND SRB- assay
 DiSC assay
 H-thymidine uptake assay
 Fluorescence
 Clonogenic Assays
In VITRO Models

8. Brine shrimp (artemia) eggs
Hatched in a hatching chamber
Containing sea water- hatched Nauplli(10 no's) pipetted
in to a vial containing suitable dilutions of the compound
vials were maintained under illumination
after 24 hrs survivors were counted.
Brine Shrimp Lethality Bioassay of
compounds

9. 
Principle: Living cell membrane has the ability to
prevent the entry of the dye, so they remain unstained
when exposed to a dye.
Ultimately, can be distinguished easily from the
Dead cells. As, the dead cells get stained.
Trypan blue exclusion assay on murine cell
lines

10. 
 It Includes two types of Assays as follows:
 MTT Assay: 3-(4, 5-dimethylthiazol-2-yl)-2, 5-
diphenyltetrazolium bromide]
 SRB Assay: SULPHORHADAMINE B
Cytotoxicity assays on panel of human
cancer cell lines

11. 
MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] taken
up by living cells
reduced to formazan by the mitochondrial enzyme succinate dehydrogenase
Formazan is a purple coloured, water-insoluble product impermeable to the
cell membranes
results in accumulation within the healthy cells, which is solubilized by DMSO
The optical density (OD) of purple coloured solution is determinedwith
ELISA plate reader at 540 nm
MTT assay Principle

12. 
 Cells from particular cell lines in log phase of growth are
trypsinised, Check the cell viability through
haemocytometer.
 Adjust to appropriate density in suitable medium and
inoculated in multiwall plates (usually 96 well micro
titer plates).
 Cells were treated with various Conc. of test compounds
and Incubated the plate at 37 °C in 5% CO 2 or 95%
humidified air (1-4 days).
 Cultures were taken out and 10 μl of MTT was added
into each well and incubated for 4hrs.
 Centrifuge the plate, discard the supernatant and
precipitated formazan salt was dissolved in DMSO.
Procedure

13. 
 The plate samples were read at 570 nm micro titer
plate reader.
 IC50 of drugs can be determined by counting the
viable cells.
 % Cell viability : {Absorbance of treated cells /
Absorbance of untreated cells}100
Continued…..

14. 
 SULPHORHADAMINE B assay measures whole
protein content which is proportional to the cell
number.
 Cell cultures are stained and staining dye is SRB
SRB Assay Principle

15. 
 SRB – a bright pink anionic protein staining dye that
binds to the basic amino acids of the cellular
proteins.
 Cell lines are counted, cultured and inoculated in 96
well plates.
 After incubation with different concentrations of test
compounds, the cell cultures are stained with SRB
dye
 Washing with CH3COOH removes the unbound dye
and the protein bounded dye is extracted using Tris
base and optical density is determined by 96-well
plate reader.
Procedure…..

16. 
 Cell suspensions are exposed to test drug
continuously for 5 days.
 Radio labelled precursor is added i.e. H- thymidine
 So replicating cells will incorporate this thymidine
into their DNA, further it is determined by
Autoradiography or Liquid Scintillation.
 Estimates: Tumor growth kinetics, Ploidy Status of
cell.
H-thymidine uptake
assay

17. 
 Cells are exposed to Fluorescent labelled precursors
after drug exposure.
 Replicating cells will incorporate this labelled
precursor into their DNA.
 Resulting fluorescence will be measured using Flow
Cytometry.
 Estimates the Actively replicating cells. And in what
phase of cell cycles cell are.
Fluorescence

18. 
 Single cell suspension are prepared from Tumor
biopsies.
 Cells are then rinsed and plated in semisolid
medium like agar.
 After 14-28 days some cells would have undergone
several divisions and formed Tumor colonies.
 Then quantitative estimates are done visually or
Semi automatically.
Clonogenic Assays

19. 
 Reduce the usage of animals.
 Testing the ability of the compound to kill the cells
by taking the advantage of various properties of cell.
 Able to process a larger number of compounds
quickly with minimum quantity.
 Range of concentrations used are comparable to that
expected for in vivo studies.
Advantages of In vitro
methods

20. 
 Difficulty in Maintaining of cultures.
 Show Negative results for the compounds which
gets activated after body metabolism and vice versa.
 Impossible to ascertain the Pharmacokinetics.
Disadvantages….

21. 
 Liquid Tumor Model using EAC cell lines
 Solid tumor model using DLA cell lines
 Xenografts
 Hollow Fiber Assay
In VIVO Models

22. 
 Albino mice are induced with Ascitic carcinoma and
further anticancer activity of the test drug is
determined.
Liquid Tumor Model using EAC cell lines

23. 
Induction of Ascitic carcinoma –
 The Ascitic tumor bearing mice (donor) were used for the
experiment 12 days after tumor transplantation.
 The Ascitic fluid was drawn using an 18 gauge needle into a
sterile syringe.
 A small amount of tumor fluid was tested for microbial
contamination.
 Tumor viability was determined by tryphan blue exclusion test
and cells were counted using haemocytometer.
 The Ascitic fluid was suitably diluted with saline to get a
concentration of 10 million cells/ml of tumor cell suspension.
 250 µl of this fluid was injected in each mouse by i.p. route to
obtain ascitic tumor.
Procedure….

24. 
 The mice were weighed on the day of tumor inoculation
and then for each three days.
 Standard drug was injected on two alternative days 1st
and 3 rd day after tumor inoculation (intraperitoneally).
 The test drugs were administered after 24 hours of tumor
inoculation and were admistered till 9th day
intraperitoneally.
 On 15th day blood was collected from the animal through
the retro orbital plexus to determine the hematological
parameters and lipid profile.
Continued…..

25. 
 % Decrease in weight variation compared to control.
 Median survival time (MST) and percentage increase in
lifespan (%ILS)
 % Increase in weight as compared to day “o”weight
 Mean survival time (MEST) and percentage increase in
lifespan (%ILS)
 Cell viability test. (% Survivors of malignant cells in
ascitic fluid)
 Hematological parameters
a. Total W.B.C. and differential leukocyte counts.
b. Total R.B.C. and Hemoglobin content.
Parameters determined

26. 
 The DLA(Dalton’s lymphoma ascitic) bearing mouse was
taken 15 days after tumor transplantation.
 The ascitic fluid was drawn using a 18 gauge needle into
a sterile syringe.
 A small amount was tested for microbial contamination
 Tumor viability was determined using Trypan blue
exclusion method and cells were counted using
haemocytometer..
 The ascitic fluid was suitably diluted in phosphate buffer
saline to get a concentration of 10 6 cells per ml of tumor
cell suspension.
Solid tumor model using
DLA cell lines

27. 
 Around 0.1ml of this solution was injected
Subcutaneously to the right hind limb of the mice to
produce solid tumor..
 Treatment was started 24 hours after tumor
inoculation.
 Standard drug was injected on two alternate days i.e.
the 1st and 3rd day.
 Extracts were administered till 9th day
intraperitoneally.
Continued….

28. 
 Tumor volume
formula : V=0.4 ab
a = major diameter
b = minor diameters respectively.
 The diameter of developing tumor with a vernier calipers
at three days interval for one month
 Tumor Weight : At the end of the fifth week animals were
sacrificed under anesthesia tumor was excised and
weighed
 % Inhibition was calculated = 1-B/A X 100
A= average tumor weight of control group
B= average tumor weight of treated group
Parameters Determined

29. 
 Small hollow fibres containing cells from human
Tumors are inserted underneath the skin or in the
body cavity.
 Test drug is administered in 2 doses, and activity is
measured.
 If the drug retards the growth of the cells then it
posses anticancer activity.
 Avg. length of the test is 4 days.
Hollow fibre Assay

30. 
 Human tumors are injected directly below the skin
of mice.
 Test drug from hollow fibre assay are given at
various dosage.
 If the test drug retard the growth of the tumor with
minimum side effects then seems to have a positive
result.
 Avg. length of the test is 30 days.
Xerographs

31. 
 https://books.co.google.in
 Resources .Guidance's and Guidelines:ICH-
http://www.fda.gov/cder/guidance/index.htm
 http://cancerres.aacrjournals.org/content/37/6/1934.ful
l.pdf
 Resources (cont’d) .Articles/Books (regulatory +
technical) DE George et al : “Regulatory considerations
for preclinical development of anticancer drugs”. Cancer
Chemotherapy Pharmacology 1998, 41: 173-185
References…

32. 
Thank you….!!