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1
“ Cancer is a disease which is characterized by
uncontrolled proliferation of cells that have
transformed from the normal ...
3
 Although 92 approved anticancer drugs are available today for the
treatment of more than 200 different tumor entities, e...
 Development of multidrug resistance in patients.
 Long-term treatment with cancer drugs is also
associated with severe ...
 INVITRO METHODS INVIVO METHODS
6
 In Vitro:
1. Tetrazolium salt assay.
2. Sulphorhodamine B assay.
3. 3H-Thymidine uptake.
4. Dye exclusion test.
5. Clono...
8
 This assay is a sensitive, quantitative and reliable
colorimetric assay that measures viability, proliferation
and activ...
 The amount of formazan produced is directly
proportional to the cell number in range of cell lines.
10
MTT Formazan
meta...
It is performed to determine the Enzymatic properties.
Cells from particular cell lines in log phase of growth are
trypsin...
After MTT dye is added in each well and plates are
incubated at 37° C for 4 hrs in a CO2 incubator.
The plates are taken o...
 Hemocytometer
The most common routine method for cell
counting which is efficient and accurate is with the use
of a hemo...
14
 % cell viability =(OD of treated cells/ OD of
control cells) × 100
The Sulphorhodamine B assay measures whole-culture
protein content, which should be proportional to the cell
number.
Cell ...
During the dead cell either lyse or are lost during
procedure, the amount of SRB binding is proportional
to the number of ...
 Large-scale, morphological changes that occur at
the cell surface, or in the cytoskeleton, can be
followed and related t...
Example: Morphological feature
(Human skin keratinocyte)
(A) (B)
Fig. Morphological feature of (A) normal human skin kerat...
Example: Morphological feature
(Human skin fibroblasts)
(A) (B)
Fig. Morphological feature of (A) normal
human skin fibrob...
 This assay is based on the structural integrity of the
cells.
 Live cells possess intact cell membranes that exclude
ce...
2. Cells were incubated with different concentrations of test
compounds for 4days.
3. Number of cultured cells in differen...
Untreated Live cells Treated cells
Tryphan Blue Dye
22
 Advantages:
• Reduce the usage of animals.
• Less time consuming, cost effective & easy to manage
• Able to process a la...
24
1. Chemical carcinogen model
DMBA induced mouse skin papillomas
 Two stage experimental carcinogenesis
› Initiator – DMBA...
Mice are topically applied a single dose of 2.5 µg DMBA in
acetone, followed by 5-10 µg of TPA in 0.2 ml acetone
twice wee...
The tumor incidence in this model is usually about 100%
DMBA controls.
In repeated topical application of DMBA Alone has a...
Mouse skin papillomas
RAT MAMMARY GLAND CA
28
 Mouse Mammary Tumor Virus (MMTV) was the first
mouse virus, isolated at Jackson labs as the “non-
chromosomal factor” th...
 Tumor cells or tissues (mouse or human) transplanted
into a host mouse.
 Ectopic – Implanted into a different organ tha...
 In vivo screening tool implemented in 1995 by NCI.
 12 human tumor cell lines (lung, breast, colon,
melanoma, ovary, an...
32
Subcutaneous Hollow Fibre implants
33
2-D and 3-D Cell-based Assays in
Drug Screening
 Currently, pharmaceutical firms spend a large amount of
money on the com...
 Currently, almost all cell-based assays or biosensors are
developed in 2-D culture systems, although conventional 2-
D c...
1.Ashish A., Sonia SY, Mark AH, and Minas TC., Pressure
Related apoptosis in Neuronal Cel Lines., Journal of
Neuroscience ...
37
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SCREENING OF ANTI CANCER DRUGS

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SCREENING OF ANTI CANCER DRUGS

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SCREENING OF ANTI CANCER DRUGS

  1. 1. 1
  2. 2. “ Cancer is a disease which is characterized by uncontrolled proliferation of cells that have transformed from the normal cells of the body. “ CAUSE:  External Factors – chemicals, radiation, viruses, and lifestyle  Internal Factors – hormones, immune conditions, and inherited mutations  Theories › Cellular change/mutation theories › Carcinogens › Oncogenes/ protooncogenes 2
  3. 3. 3
  4. 4.  Although 92 approved anticancer drugs are available today for the treatment of more than 200 different tumor entities, effective therapies for most of these tumors are lacking.  Out of the 92 registered drugs, 17 are considered by oncologists to be more broadly applicable and 12 additional agents are perceived as having certain advantages in some clinical settings  They are mostly cytotoxic in nature and act by a very limited number of molecular mechanisms.  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. 4
  5. 5.  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 5
  6. 6.  INVITRO METHODS INVIVO METHODS 6
  7. 7.  In Vitro: 1. Tetrazolium salt assay. 2. Sulphorhodamine B assay. 3. 3H-Thymidine uptake. 4. Dye exclusion test. 5. Clonogenic test. 6. Cell counting assay. 7.Morphological assay  In Vivo: 1. Carcinogen induced models 2. Viral infection models 3. Transplantation Models 4. Genetically Engineered Mouse Models 5. In vivo hollow fibre assay 7
  8. 8. 8
  9. 9.  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-2- yl)-2,5-diphenyl tetrazolium bromide (MTT) into a dark blue formazan product which is insoluble in water. 9
  10. 10.  The amount of formazan produced is directly proportional to the cell number in range of cell lines. 10 MTT Formazan metabolically active Cell Insoluble
  11. 11. 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 11 METHOD:
  12. 12. After MTT dye is added in each well and plates are incubated at 37° C for 4 hrs in a CO2 incubator. The plates are taken out from the incubator and dark- blue colored formazan crystal are thoroughly dissolved in DMSO in room temperature. The plates are then read on a ELISA reader at 570 nm To calculate the percent cell viability with respect to control is calculated . 12
  13. 13.  Hemocytometer The most common routine method for cell counting which is efficient and accurate is with the use of a hemocytometer. 13
  14. 14. 14  % cell viability =(OD of treated cells/ OD of control cells) × 100
  15. 15. 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, Sulphorhodamine B. 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. 15
  16. 16. 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. 16
  17. 17.  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 17
  18. 18. Example: Morphological feature (Human skin keratinocyte) (A) (B) Fig. Morphological feature of (A) normal human skin keratinocyte, and differentiated human skin keratinocyte(B). 18
  19. 19. Example: Morphological feature (Human skin fibroblasts) (A) (B) Fig. Morphological feature of (A) normal human skin fibroblasts, and aging human skin fibroblasts(B). 19
  20. 20.  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. METHOD: 1. Cell lines are counted, cultured and innoculated in 96 well plates as above. 20
  21. 21. 2. Cells were incubated with different concentrations of test compounds for 4days. 3. 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 (viable+dead) ×100 21
  22. 22. Untreated Live cells Treated cells Tryphan Blue Dye 22
  23. 23.  Advantages: • Reduce the usage of animals. • Less time consuming, cost effective & easy to manage • 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.  Disadvantages: • 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 23
  24. 24. 24
  25. 25. 1. Chemical carcinogen model DMBA induced mouse skin papillomas  Two stage experimental carcinogenesis › Initiator – DMBA (dimethylbenz[a]anthracene), › Promotor – TPA (12-O-tetradecanoyl-phorbol-13- acetate)  Mice : Single dose – 2.5 µg of DMBA , 5 to 10 μg of TPA in 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 25
  26. 26. Mice are topically applied a single dose of 2.5 µg DMBA in acetone, followed by 5-10 µg of TPA in 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 or oral route. 26
  27. 27. The tumor incidence in this model is usually about 100% DMBA controls. In repeated topical application of DMBA Alone has also been shown to induced carcinogenesis. 27 Drug efficacy is measured as percent reduction in carcinoma incidence, compared with that of carcinogen control.
  28. 28. Mouse skin papillomas RAT MAMMARY GLAND CA 28
  29. 29.  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 random integration in certain cells  Some viruses carry cellular oncogenes › Abelson murine leukemia virus – Abl › Moloneymurine sarcoma virus – Raf  Engineered viruses now used routinely in the laboratory to induce cancer. 29
  30. 30.  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 30
  31. 31.  In vivo screening tool implemented in 1995 by NCI.  12 human tumor cell lines (lung, breast, colon, melanoma, ovary, and glioma.  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 31
  32. 32. 32
  33. 33. Subcutaneous Hollow Fibre implants 33
  34. 34. 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 to developing 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. 34
  35. 35.  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. 35
  36. 36. 1.Ashish A., Sonia SY, Mark AH, and Minas TC., Pressure Related apoptosis in Neuronal Cel Lines., Journal of Neuroscience Research 2000 60: 495-503 2.Essentials of medical pharmacology ,by KD Tripathi, 7th edition ,page no :857 3.https://www.slideshare.net/ashwinisomayaji7/screening- of-anticancer-drugs 4.https://en.wikipedia.org/wiki/Chemotherapy. 5. http://www.embl-heidelberg.de/ ExternalInfo/karsenti/countingcells. html 36
  37. 37. 37

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