Mattingly "AI & Prompt Design: The Basics of Prompt Design"
PHARMACOLOGY OF ANTICANCER DRUGS.pptx
1.
2. MAHARSHI DAYANAND UNIVERSITY
Department Of Pharmaceutical Science
PHARMACOLOGICAL & TOXICOLOGICAL SCREENING
MODELS-1
PRECLINICAL SCREENING MODELS OFANTICANCER DRUGS
SUBMITTED BY: UNDER THE GUIDANCEOF:
DR. ABHILASAHA AHLA
W
AT
ASSISTANT PROFESSOR OF
MDU ROHTAK.
TARANJUM KHAN
M.PHARMACY IST YR
PHARMACOLOGY
ROLLNO: 1803
4. OUTLINES
▶ Introduction.
▶ Factors
▶ Novel Need of Anticancer Drugs
▶ Preclinical screening models
▶ Detail study Of Invitro Models
▶ Detail study Of Invivo Models
▶ Summary
▶ References
5. Introduction:
▶ Cancer is a disease which is characterizedby uncontrolledproliferationof cells that have transformed
from the normal cells of the
▶ Induction of proto-oncogenes and inhibition of tumor suppressorgenes has been implicated in the
pathogenesis of cancer
▶ Causes
▶ External Factors - chemicals, radiation, viruses, and lifestyle.
▶ Internal factor- hormones, immune conditions, and inherited mutations
▶ Types of tumors
▶ Benign
▶ PremalignantAnd Malignant
6. Factors Believed to Contribute to Global
Causes of Cancer
▶ Although 92 approved anti cancer drugs are available
today for the treatment of more than 200 different
tumor entities, effective therapies for most of these
tumors are lacking.
▶ Thus, the need for novel drugs to treat malignant
disease requiring systemic therapy is still pressing
▶ A preselection, called the screening process, is therefore
required.
▶ The aim of screening efforts is to identify products that
will produce antitumor effects matching the activity
criteria used to define which compounds can progress
to the next stage in the preclinical development
program.
7. Need For Novel Anticancer Drugs
▶ 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
▶ Cancer is the second leading cause of death globally, responsible for an estimated
9.6 million deaths in 2018, and this burden continues to increase. Therefore, there
is a clear and urgent need for novel drugs with increased efficacy for the
treatment of different cancers.
10. Invitro Models
▶ Advantages:
▶ Reduce the usage of animals.
▶ Less time consuming,Cost effective & Easy to
manage
▶ A controlledenvironment can be
maintainedAble to processa larger number of
compoundsquickly with minimum quantity.
▶ Disadvantages
▶ maintaining of cultures.
▶ Show false positive and show negative results
for the compoundswhich gets activated after
body metabolism
11. 1. Tetrazolium Salt Assay
▶ This assay is a sensitive, quantitative and reliable
colorimetric assay that measures viability,
proliferation and activation of cells.
▶ The assay is based on the capacity of
mitochondrial dehydrogenase enzymes in living
cells to convert the yellow water-soluble substrate
3-(4,5-dimethylthiazol-2yD)-2,5-diphenyl
tetrazolium bromide (MTT) into a dark blue
formazan product which is insoluble in water.
▶ The amount of formazan produced is directly
proportional to the cell number in range of cell
lines.
12. Method:
▶ It is performed to determine the Enzymatic properties.
▶ Cells from particular cell lines in log phase of growth are trypsinised
▶ It is counted in a hemocytometer and adjusted multiwell plates(96 well plates
▶ The cells are treated with a various concentration of drug for specified duration
▶ After MTT dye is added in each well and plates are incubated at 37° C for 4 hrs in a Co,
incubator.
▶ The plates are taken out from ne incubator and dark blue colored formazan crystal are
thoroughly dissolved in DMSO in room temperature.
▶ The plates are then read with ELISAreader at 570 nm
▶ To calculate the percent cell viability with respect to control is calculated.
13. 2.Hemocytometer Cell Counts:
▶ The most common routine method for cell countingwhich is efficient and accurate is with the use of a
hemocytometer.
▶ O % cell viability =(OD of treated cells/ OD of controlcells)× 100
14. 3.Sulphorhodamine B Assay :
▶ The Sulphorhodamine B assay measures whole-culture protein content,
which should be proportional to the cell number.
▶ Cell culture are stained with a protein staining dye, SulphorhodamineB.
▶ SRB is a bright pink anionic dye that binds to basic amino acid of cell.
Unbound dye is then removed by washing with acetic acid.
▶ During the dead cell either lyse or are lost during procedure, the amount
of SRB binding is proportional to the number of live cells left in a culture
after drug exposure.
15. Colonogenic Assay:
▶ Invitro cell survival assay based on the ability of single cell to grow into a colony
▶ Assay tests every cells in population for its ability to undergo unlimited division
▶ Imp. for drugs act by arresting check points in cell cycle.
▶ Drug efficacy:
▶ % reduction in adenoma incidence, multiplicity or % increase in adenoma latency compared
with that of carcinogen control
▶ Tumor multiplicity: 2-4
▶ Tumor latency: 65-80 days
16. Morphological Assay:
▶ Large-scale, morphological changes that occur at the cell surface, or in the
cytoskeleton, can be followed and related to cell viability.
▶ Damage can be identified by large decreases in volume secondary to losses in
protein and intracellular ions due to altered permeability to sodium or potassium.
▶ Necrotic cells: nuclear swelling, chromatin flocculation, loss of nuclear
basophilia
▶ Apoptotic cells: cell shrinkage, nuclear condensation, nuclear fragmentation.
18. Dye Exclusion Test:
▶ This assay is based on the structural integrity of the cells.Live cells possess intact cell membranes that
exclude certain dyes, such as tryphan blue, Eosin, or propidium, whereas dead cells would have lost
membrane integrity.
▶ Hence they would take up the dyes while the live cells exclude it.
▶ To determine cell viability in which dilutesolution of dyes (trypan blue, eosin Y, nigrosin,Alcian blue) is
mixed with suspensions of cells
▶ Cells excludedye :- alive
▶ Cells that stain:- dead
▶ Indicate structural integrity of cell membrane
▶ Wiesenthaland colleagues used novel combination of fast green dye & eosin - hematoxylin with more
promising results, in patients with hematological malignancies (CLL).
19. Method:
▶ Cell lines are counted, cultured and inoculated in 96 well plates as above.
▶ Cells were incubated with different concentrations of test compounds for 4days.
▶ Number of cultured cells in different wells were counted using hemocytometer
after staining with suitable dyes.
▶% cell viability = no. of viable cell Total no.of cells X100
21. In vivo Methods
▶ Advantages:
▶ Detect host mediated activity
▶ Relatively predictable Estimate therapeutic ratio
▶ Used for both preclinicalanticancerefficacy
detectionand for toxicological studies.
▶ Disadvantages:
▶ Sensitivity is low
▶ Costly,Time consuming
▶ Large number of samples cannot be handled.
▶ Difficult to manage
22. In vivo Models:
Chemically Induced Tumor
Model
▶ DMBA-inducedmouse skinpapillomasrat
mammary gland carcinogenesis, oral cancer in
hamster.
▶ MNU-inducedrat mammary gland
carcinogenesis,tracheal sq cell CAin hamsters,
prostatecancerin gerbils
▶ DEN-inducedlung adenoCAin hamsterDMH-
induced colorectaladenoCAin rat and mouse.
▶ OH-BBN induced bladderCAin
mouseHepatocellulerCAmodels
Method Involving Cell line
Implantation
▶ Cell line implantationHollowfiber techniqueUse of
Xenografts
▶ Nude mouse model
▶ Newborn rat model &Transgenic mouse model
▶ Viral infectionmodels:Mousemammary tumor
virus
▶ Moloney murine sarcoma virus
▶ Newer geneticallyengineered viruses
23. Chemical Carcinogen Models
▶ DMBA induced mouse skin papillomas
▶ Two stage experimental carcinogenesis
▶ Initiator - DMBA (dimethylbenzajanthracene)
▶ Promotor - TPA (12-O-tetradecanoyl-phorbol-13 acetate).
▶ Mice: Single dose 2.5 ug of DMBA, 5 to 10 ug of TPAin 0.2 ml of acetone twice
weekly.
▶ Papilloma begins to appear after 8 to 10 wks Tumor incidence & - multiplicity of
treatment group is compared with DMBA control group
24. Method:
▶ Mice are topically applied a single dose of 2.5 ug DMBA in acetone, followed by 5-10 ug of
TPAin 0.2 ml acetone twice weekly on the same site starting one week after DMBA
application.
▶ Percent tumor incidence and multiplicity of treatment groups is compared with DMBA
control group.
▶ Drug under test can be administered either topically oral route.
▶ The tumor incidence in this model is usually about 100% DMBA controls.
▶ In repeated topical application of DMBAAlone has also been shown to induced
carcinogenesis.
▶ Drug efficacy is measured as percent reduction in carcinoma incidence, compared with that
of carcinogen control.
25.
26. DMBA-induced Rat Mammary Gland
Carcinogenic
▶ Female Sprague-Dawley are use for this method.
▶ Rats are given single intragastric injection of 12 mg/kg DMBA at 50 days of age.
▶ This dose results in 80-100% incidence of total mammary tumors within 120 days
post carcinogen.
▶ This model can detect the agents/drugs inhibiting carcinogen activation.
▶ DNBA produced capsulated tumors with high incidence.
▶ Drug efficacy is measured as percent reduction in adenoma incidence, percent
increase in adenocarcinoma latency compared with that of carcinogen control.
27. Viral Infections Models
▶ Mouse Mammary Tumor Virus (MMTV) was the first mouse virus, isolated at Jackson labs as the
"non chromosomal factor" that caused mammary tumors in the C3H strain of mice.
▶ Some viruses cause cancer via randomintegration in certain cells
▶ Some viruses carry cellularoncogenes
▶ Abelson murine leukemia virus – Abl
▶ Moloneymurinesarcoma virus - RafEngineered viruses now used routinelyin the laboratoryto induce
cancer.
28. 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-
proceed to next stage of testing
▶ 1. Spheroid culture of LuCap 147-induced prostate cancer model
▶2.Integration free-induced pluripotent stem cell modelhigh throughput screeningdrug
induced cell cycle arrestApoptosiscan be demonstrated in spheroid cultures
29. Straining Model Of laboratory Animals
▶ NEWBORN RAT MODEL
▶ Can be used as an alternative to nude
mouse
▶ As cost effective and maintenance is easy
▶ NUDE MOUSE:
▶ Immunologically incompetentmouse due to
absence of thymus
▶ Do not show contact sensitivity or reject the
transplant material
▶ Melanomas, colon carcinomas grow very well
30. Hepataocellular Carcinoma Model
▶ Can be readily induced by chemical carcinogens
▶ Several animal models are well established
▶ Naturally occuring- Wood chuck, Long Evans Cinnamon rat
▶ 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.
31. Transplantation Models
▶ Tumor cells or tissues (mouse or human) transplanted into a host mouse.
▶ Ectopic Implanted into a different organ than the original (typically subcutaneous or
kidney capsule)
▶ Orthotopic Implanted into the - analogous organ of the original tumor.
▶ Advantages:
▶ Typically cheap, fast & easy to use.
▶ Not covered by patents
32. In-Vivo Hollow Fiber Assay
▶ In vivo screening tool implemented in 1995 by NCI.
▶ 12 human tumor cell lines (lung, breast, colon, melanoma, ovary, andglioma.
▶Cells suspended into hollow polyvinylidene fluoride fibers implanted IP or SC
in lab mice.
▶ After in vivo drug treatment, fibers are removed and analyzed in vitro.
▶ Antitumor (growth inhibitory) activity assessed
35. 2-D and 3-D Cell-based Assays in Drug
Screening
▶ Currently, pharmaceutical firms spend a large amount of money on the compound
efficacy and cytotoxicity test.
▶ There is still a 78% failure rate for all drugs, which may be devastating todeveloping
companies.
▶ Effective compounds in vitro may be non-effective in vivo for many reasons,
including differences between in vitro and in vivo target biology, interrelated
biochemical mechanism, metabolism, poor penetration into solid tissues, etc.
▶Currently, almost all cell-based assays or biosensors are developed in 2-D culture
systems, although conventional 2 D cultures usually suffer from contact inhibition
and a loss of native cell morphology and functionality.
36. Continue….
▶ Currently, almost all cell-based assays or biosensors are developed in 2-D culture systems, although
conventional 2 D cultures usually suffer from contact inhibition and a loss of native cell morphology and
functionality.
▶ In comparison with 2-D cultures, 3-D cell models create a more realistic representation of real human
tissues, which is critical to many important cell functions, including morphogenesis, cell metabolism, gene
expression, differentiation and cell-cell interactions
37.
38. EAC Cells by Liquid Tumour Model (Ehrlich
Ascites Carcinoma) on (Swiss albino mice)
▶ EXPERIMENTAL PROTOCOL
▶ Induction of ascitic carcinoma :
▶The ascitic tumor bearing mice (donor) were used. 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
39. Continue…….
▶ The mice were weighed on the day of tumor inoculationand then for each three days.
▶ Cisplatin was injected on two alternative days 1st and 3rd day after tumor inoculation(intraperitoneally).
▶ The drugs were administered after 24 hours of tumor inoculationand were admistered till 9th day
intraperitoneally.
▶ On 15th day blood was collected from the animal through the retroorbitalplexus to determine the
heamatological parameters and lipid profile
▶ Parameters monitored
▶ 1) % Decrease in weight variationcompared to control.
▶ 2) Median survivaltime (MST) and percentageincreasein lifespan (%ILS).
▶ 3) Mean survivaltime (MEST) and percentage increasein lifespan (%ILS).
▶ 4) Cell viabilitytest.
40. ▶ % Increase in weight as compared to day "0"weight Upon weighing the animals on
the day of inoculation and after once in 3 days in the post inoculation period the %
increase in weight was calculated as follows
▶ Median survival time and increase in life span [%ILS]Total number of days an animal
survived from the day of tumor inoculation was counted. Subsequently the Median
and Mean survival times were calculated. The %ILS was calculated as follows.
41. References
▶ AshishA., Sonia SY, MarkAH, and Minas TC., Pressure Related apoptosis in Neuronal Cell Lines.,
Journal of Neuroscience Research 2000 60: 495-5032.
▶ KD Tripathi, Essentials of medical pharmacology, 7th edition page no :853.
▶ http://www.emblheidelberg.de/ExternalInfo/karsenti/countingcells.html
▶ Rubinstein LV, Shoemaker RH, Paull KD, Simon RM, Tosini S, Skehan P, et al. Comparison of in
vitro anticancer-drug-screening data generated with a tetrazolium assay versus a protein assay
against a diverse panel of human tumor cell lines
▶ .Vistica DT, Skehan P, Scudiero D, MonksA, PittmanA, Boyd MR. Tetrazolium-basedassays for
cellular viability:A critical examination of selected parameters affecting formazan
production. Cancer Res. 1991;51:2515
▶ kehan P, StorengR, Scudiero D, MonksA, McMahon J, Vistica D, et al. New colorimetric
cytotoxicity assay for anticancer-drugscreening. J Natl Cancer Inst. 1990;82:1107–12.
▶ Papazisis KT, Geromichalos GD, Dimitriadis KA, KortsarisAH. Optimization of the sulforhodamine
B colorimetric assay. J Immunol Methods. 1997;208:151–8