Post on 09-Jan-2017
Preclinical Trials for Anticancer
drugsPriyanka Sanas
Master of Pharmacy: Pharmacology1st Year
What is Cancer Tumors Types Of Cancer Need for Novel Anticancer Agents In Vitro Models In VIVO Models
Contents..
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…???
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 Tumor2. MALIGNANT Tumor
Tumors
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
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
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
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
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
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
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
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
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…..
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
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…..
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
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
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
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
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….
Liquid Tumor Model using EAC cell lines Solid tumor model using DLA cell lines Xenografts Hollow Fiber Assay
In VIVO Models
Albino mice are induced with Ascitic
carcinoma and further anticancer activity of the test drug is determined.
Liquid Tumor Model using EAC cell lines
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….
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…..
% 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
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
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….
Tumor volume
formula : V=0.4 ab a = major diameterb = 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 100A= average tumor weight of control groupB= average tumor weight of treated group
Parameters Determined
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
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
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.full.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…
Thank you….!!