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….!!