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Preclinical and clinical screening of drugs for anticancer activity
1. Preclinical and clinical screening of drugs
for anticancer activity (solid tumors)
Dr. Banhisikha Adhikari
2. OUTLINE
⢠Introduction
⢠Preclinical screening
In vitro methods
In vivo methods
⢠Microdosing
⢠Newer Approaches
⢠Summary
⢠Clinical Screening
Phase I
Phase II
Phase III
Phase IV
3. INTRODUCTION
⢠Cancer is a disease characterized by uncontrolled
proliferation abnormally transformed cells
⢠There are more than 100 types of cancer
⢠A multifactorial disease
⢠Induction of proto-oncogenes and inhibition of tumor
suppressor genes has been implicated in the
pathogenesis of cancer
⢠Anticancer drugs are developed from variety of
sources ranging from natural products to synthetic
molecules
4. Drugs widely used as cancer chemotherapeutic agent suffers
from drawbacks like â
ď High toxicity(bone marrow suppression, alopecia, nausea,
risk of secondary cancers)
ď High cost
ď Development of resistance
ď Less tumor cell selectivity
ď Necessiates development of compounds with lesser
toxicity, tumor cell selectivity, novel targets and more cost
effective
ď For this quick and novel methods are being identified that
can screen a large number of compounds
ď In vitro and in vivo models are systematically applied for
screening of anticancer drugs
6. In Vitro Methods
Advantages
⢠Reduce the usage of
animals.
⢠Less time consuming,
⢠Cost effective &
⢠Easy to manage
⢠A controlled environment
can be maintained
⢠Able to process a larger
number of compounds
quickly with minimum
quantity.
Disadvantages:
⢠Difficulty in
maintaining of
cultures
⢠Show false positive
results
⢠Show negative results
for the compounds
which gets activated
after body
metabolism
⢠Impossible to
ascertain the
Pharmacokinetics
7. ⢠The goal of screening assay is to test ability of a compound
to kill cells
⢠Should be able to discriminate between replicating and
nonreplicating cell
⢠Different assays take advantage of various properties of cell
as mentioned below
CELL PROPERTIES ASSAY
ENZYMATIC PROPERTY MTT ASSAY
PROTEIN CONTENT SRB(SULPHORHODAMINE B) ASSAY
DNA CONTENT/REPLICATION STATUS 3H-THYMIDINE UPTAKE & FLUORESCENT
ASSAY
MEMBRANE INTEGRITY DYE EXCLUSION TEST
COLONY FORMING POTENTIAL CLONOGENIC ASSAY
CELL DIVISION CELL COUNTING ASSAY
8. Microculture Tetrazolium Test(MTT)
⢠A quantitative colorimetric assay
⢠Measures cellular growth, cell
survival and cell proliferation
⢠Yellow Dimethyl thiazol
diphenyltetrazolium bromide a
tetrazolium salt is reduced to purple
formazan by mitochondrial
dehydrogenase of living cells
⢠Intensity of formazan produced is
directly proportional to cell viability
9. Cells from particular
cell line at log phase
of growth
tryptanised, counted
in haemocytometer,
adjusted to
appropriate density
Inoculated in
different mutiwell
plates, treated with
various conc of drugs
for specified
duration
MTT dye is added
and incubated at
37°C for 4 H in a
CO2 incubator
The percent of cell viability
with respect to control is
calculated using the formula
%cell viability=
(OD of treated cell/OD of
control cell)Ă 100
Plates are read
on an ELISA
reader at
570nm
Taken out of
incubator purple
coloured formazan
formed thoroughly
mixed with
isopropanol/DMSO
10. Sulphorhodamine B Assay
⢠Measures whole culture protein content
⢠SRB is a bright pink anionic protein staining dye that
binds to basic amino acid of cell.
⢠Cell cultures are stained with it and unbound dye is
removed by washing with acetic acid
⢠Determination of optical density in a computer
interfaced,96-well micro titer plate reader
⢠The amount of SRB binding is proportional to the
number of live cells left in a culture after drug
administration
⢠Screening capacity, reproducibility, quality control all
appear to be enhanced
11. 3H-thymidine uptake assay:
⢠Replicating cells will incorporate 3H-thymidine which then can be
determined by autoradiography or liquid scintillation counting
⢠Provides information on tumor growth kinetics
⢠DNA Histogram-information on ploidy status of cell
Fluoroscence:
⢠Fluorescent dye incorporation followed by microcsopic evaluation
⢠Replicating cells incorporate labeled precursor into their DNA and
resulting Fluoresence is measured by flow cytometry
Dye Exclusion Test:
⢠Relied on structural integrity of cell.
⢠Dead cells have lost membrane integrity and would take up vital dyes
like tryptan blue
⢠Modified version-Differential staining cytotoxicity assay
⢠End point morphologic identification of tumor cell cytotoxicity
compared with internal control
12. Clonogenic assays:
⢠Measure tumor cell reproductive viability
⢠Most direct method of measuring cytotoxicity of a drug
Cell counting assay:
⢠Cells are cultured in the presence of drugs for 2-5 culture doubling
times
⢠Cell number is estimated using a haemocytometer or cell counter
3D Tumor Models:
⢠Cancer cells are cultured in a spatially relevant manner with
endothelial cells and other cells
⢠Biomimetic property accurately depict in vivo situation of drug
screening
⢠Advantageous over complexity of animal models and the spatial
limitaion of cell culture model
4D Tumor Model:
Ex vivo lung cancer model of perfusable nodule on a lung matrix
13. Examples of 3D models
Spheroid system:
⢠Three dimensional multicellular tumors derived from HeLa cell
⢠Quantify chemotherapeutic and nanoparticle penetration in
vitro
⢠Acquire several clinically relevant morphologic and cellular
characteristic often found in human solid tumors
Spherochip system:
⢠Automated assay
⢠Microfluidic based platform for long term 3D cell culture
⢠Analysis is compatible with commercially available microplate
⢠readers
⢠Dynamic change in metabolic activity of cell can be observed
14. In Vivo methods
⢠Aimed at predicting
Safe starting dose & dosage regimen for human clinical trials
The toxicities of the compound, &
The likely severity and reversibility of drug toxicities
Advantages:
⢠Detect host mediated
activity
⢠Relatively predictable
⢠Estimate therapeutic
ratio
⢠Used for both
preclinical anticancer
efficacy detection and
for
toxicological studies
Disadvantages:
⢠Sensitivity is low
⢠Costly
⢠Time consuming
⢠Large number of
samples cannot be
handled
⢠Difficult to manage
15. Chemically Induced tumor
models:
⢠DMBA-induced mouse skin
papillomas, rat mammary
gland carcinogenesis, oral
cancer in hamster
⢠MNU-induced rat
mammary gland
carcinogenesis, tracheal sq
cell CA in hamsters,
prostate cancer in gerbils
⢠DEN-induced lung
adenoCA in hamster
⢠DMH-induced colorectal
adenoCA in rat and mouse
⢠OH-BBN induced bladder
CA in mouse
⢠Hepatocelluler CA models
Models involving cell
line/tumor pieces
implantation:
⢠Cell line implantation
⢠Hollow fiber technique
⢠Use of Xenografts
⢠Nude mouse model
⢠Newborn rat model
⢠Transgenic mouse
model
Viral infection models:
⢠Mouse mammary
tumor virus
⢠Moloney murine
sarcoma virus
⢠Newer genetically
engineered viruses
16. DMBA-induced Mouse skin papilloma
⢠classical two stage experimental
carcinogenesis model
⢠SENCAR mouse is highly sensitive-tumor
incidence 100% in controls
⢠Dimethylbenzanthracene(DMBA) acts as
initiator and tetradecanoyl phorbol
acetate is used as promoter
⢠Topically applied on shaved back till
appearance of papillomas (6-7) weeks
⢠Weekly monitoring of tumor development
for 18 weeks
⢠Percent tumor incidence and multiplicity
are compared between treatment and
DMBA control groups.
Mouse skin papilloma
17. MNU induced rat mammary gland CA
⢠Induces hormone dependent
tumors.
⢠Single i.v of 50 mg/kg of
methylnitrosourea(MNU) given to
50 days old Sprague-Dawley rats.
⢠Adenocarcinoma will be produced
within 180 days of post carcinogen
in 75 to 95% cases
⢠Drug efficacy is measured
⢠Drawback â cannot detect inhibition
of carcinogen activation
18. Other similar models
Cancer site Cancer Type Species Carcinogen
Colon Adenocarcinomas Rat, Mouse AOM (azoxymethane),
DMH
(Dimethylhydralazine)
Prostate Adenocarcinomas Gerbil MNU (methylnitrosourea)
Trachea Squamous cell
carcinoma
Hamster MNU (methylnitrosourea)
Breast Adenocarcinoma Mouse NMU (methylnitrosourea)
DMBA
(Dimethylbenzanthracene)
Lung Adenocarcinoma Hamster DEN (Diethylnitrosoamine)
19. Hepatocellular carcinoma models
⢠Can be readily induced by chemical carcinogens
⢠Several animal models are well established
⢠Naturally occuring- Wood chuck, Long Evans Cinnamon
rats
⢠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.
20. Methods involving cell line:
⢠Specified number of particular cell line inoculated into sensitive mouse
strain
⢠Tumors develop rapidly thus time saving
⢠Effective drug retard tumor growth and increase life span of animal
⢠L-1210, P-388, B-16 cell lines- host mouse strain BDF1
⢠Sarcoma-180 â Swiss albino mouse
Hollow Fiber Technique:
⢠Small hollow fibers containing cells
from human tumors
⢠Inserted underneath skin and in body
cavity of mouse
⢠Candidate drug tested in vivo
⢠Compounds retarding growth
are recommended for next level of testing
Subcutaneous hollow fiber implant
21. 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-procced to next stage of testing
⢠1. Spheroid culture of LuCap 147-induced prostate cancer model
2.Integration free-induced pluripotent stem cell
model
high throughput screening
drug induced cell cycle arrest
apoptosis
can be demonstrated in spheroid cultures
22. Nude mouse:
⢠Immunologically incompetent mouse due to absence of thymus
⢠Do not show contact sensitivity
or reject the transplant material
⢠Melanomas, colon carcinomas
grow very well
Newborn rat model:
⢠Can be used as an alternative to nude mouse
⢠as cost effective and maintenance is easy
Transgenic mouse model:
⢠Inactivation of a particular gene within specific tissues of adult mouse
⢠Serve as both model of disease as well as gene therapy
⢠Metamouse: tumor pieces of patients are directly transplanted to
organ of primary growth
⢠Metastasis and weight loss occurs same way as in humans
⢠Test new routes, doses and indications of old drugs
24. PHASES
⢠Phase I: to identify safe dose levels and schedules
⢠Phase II: to identify the spectrum of anticancer
activity
⢠phase III: to compare the New Chemical Entity (NCE)
with the up-to-then best-available treatment
⢠phase IV: continue to monitor drug safety as it is
then administered to a significantly greater number
of patients
⢠Frequently drug combinations are evaluated instead
of a single compound monotherapy
25. Phase I
⢠Phase I studies of anticancer agents are usually
conducted in patient
⢠Carried out at progressively escalating doses to identify
the dose-limiting toxicities for cytotoxic compounds
⢠Increments in drug doses : based on the type, severity,
and duration of observed toxicities
⢠Concludes when the Maximum Tolerated Dose is reached
⢠Necessary information on the clinical toxicity,
pharmacokinetics, and preliminary antitumor activity are
gathered
26. ⢠Challenges:
(1) recruitment of tumor-specific patient volunteers becomes
difficult
(2) the recruited volunteers are usually in the advanced stages
of the disease and refractory to the currently available
standard-of- care treatment options
Takes into account:
Dose escalation
Inter-patient Variability and Dose Normalization
Drug Combinations and Dosing Strategies
Adverse Effects and Toxicities of Anticancer Drugs
Special Patient Populations
27. Phase II
⢠Done in a small group of patients with a specific tumor type to
determine anticancer efficacy and to define the therapeutic
window
⢠Traditionally designed as single-arm trials utilizing historical
controls
⢠Use the proportion of patients who achieve a complete or partial
response to the treatment as the primary efficacy measure
⢠To avoid exposing patients to inactive compounds statistical tools
are used to interrupt studies where the in-process data indicate
low probability of success
⢠Act as a screen of antitumor efficacy to select the most promising
agents to enter the pivotal phase III clinical trials.
28. Phase III
⢠Are conducted in a much greater number of patient volunteers of
the selected tumor type
⢠Usually use a parallel-arm design
⢠Prospective and randomized evaluation against the then-available
best-possible therapy for the disease
⢠The demonstration of statistically significant improvement in tumor
response against the currently best-available treatment in a tumor
type specific patient population ultimate benchmark for
regulatory approval and marketing of a novel anticancer agent
⢠Usually conducted by certain cooperative groups:
Eastern Cooperative Oncology Group
Childrenâs Oncology Group
Cancer and Leukemia Group
29. Trial features
Include a clear definition of the
⢠objectives
⢠end points
⢠inclusion and exclusion criteria
⢠treatment plan
⢠clinical assessments
⢠laboratory tests
⢠trial design(including
randomization)
⢠statistical considerations
⢠data monitoring protocols
⢠and informed consent
⢠Blinding is often not
utilized: because of distinct
dosing schedules, routes of
administration, and toxicity
profiles
⢠often non-inferiority trials
are conducted with the
goal to prove that the
therapeutic benefit of a
drug is not lost with a new
regimen or treatment
approach
⢠Crossover designs are not
preferred
30. End Points
⢠End point for determining the efficacy in clinical trials of anticancer
drugs is an evolving subject
⢠Phase III cancer clinical trials - one primary end point of clinical
efficacy and one or more secondary end points like reduced side
effects
⢠Three kinds of end points have been used:
(1) objective tumor response, e.g., size regression
(2) time to event end points
(3) patient-reported outcomes like palliation of side effects
⢠The newer molecularly targeted anticancer agents have different
measurement of efficacy evaluation
⢠Determination of clinical end points for these drugs
quantifiable pharmacodynamic characteristics like target inhibition
or the levels of a tumor-specific biochemical marker
31. Tumor Regression End points:
⢠Complete response
⢠Partial response
⢠Stable disease or
⢠Progressive disease
Time to event end points:
⢠Overall survival (OS)
⢠Disease-free survival (DFS)
⢠Time to progression (TTP)
⢠Time to treatment failure (TTF)
⢠Progression free survival (PFS)
32. Microdosing in cancer trials
⢠The first-in-human clinical trials of novel compounds constitute a
significant safety risk for the patient volunteers.
A microdosing strategy
⢠Mitigate risk
⢠Gather pharmacokinetic data
⢠More accurately predict the first-in-human doses
⢠The microdose of a small-molecule drug less than 1/100th of
the dose calculated to yield a pharmacological effect
⢠Protein drug 30 nmol is considered the maximum dose
⢠complement the existing animal-to-human dose-scaling strategies
⢠Unable to predict PK parameters where drugs exhibit nonlinear
pharmacokinetics
33. Newer Approaches
⢠Active immunotherapies: cancer vaccines stimulate a patientâs
immune system to destroy cancer cellsâ sipuleucel-T(Provenge)
⢠Liposomes: Liposomes are drug-delivery vesicles with a lipid
bilayer enclosing an aqueous solution. Eg- liposomal daunorubicin
(DaunoXome), liposomal doxorubicin (Myocet), and liposome-PEG
doxorubicin (Doxil/Caelyx)
⢠Prodrugs: Prodrugs that are specifically activated at the tumor site
will reduce systemic toxicity and increase efficacy
⢠Delivering drugs differently: Delivery systems tagged with
mAbs to direct a therapeutic straight to the cancer cells and with
imaging agents to track drug delivery and disease progress
⢠Overcoming resistance: Cellular pathways and genes that may
confer resistance are used as drug targets
⢠Personalized medicine: Gene-expression diagnostics and
biomarkers are used to stratify patients into potential responders
and nonresponders
34.
35. Summary
⢠Focus has shifted from cytotoxic compounds to target
based therapy
⢠Basic research in cancer biology has provided new targets
for drug development and brought older targets to
sharper focus
⢠Newer treatment regimen, routes of administration,
approaches are increasingly evaluated
⢠Current challenges are significant time and cost
involvement and low success rate
⢠For this faster and more predictable screening methods
are being developed
⢠Continuous improvement now is incorporating a risk
based approach
Clinical trials of drug candidates are carried out in three distinct phases: phase I
studies to identify safe dose levels and schedules, phase II studies to identify the
spectrum of anticancer activity, and phase III studies to compare the NCE with
the up-to-then best-available treatment. In addition, post-marketing surveillance
phase IV studies continue to monitor drug safety as it is then administered
to a significantly greater number of patients. Regulatory involvement is critical
at all stages of clinical drug development. As illustrated in Fig. 2, an Investigational
New Drug (IND) application is filed with the U.S. Food and Drug
Administration (FDA) before the initiation of phase I studies. At the end of
phase II studies, usually a pre-NDA meeting is held with the FDA to discuss
the results and the plans for the phase III clinical trials. Upon completion of the
phase III studies, a New Drug Application (NDA) is filed with the FDA for the
grant of marketing authorization.
The first-in-human clinical trials of novel cytotoxic compounds constitute a
significant safety risk for the patient volunteers. A microdosing strategy has
been proposed to mitigate this risk, gather pharmacokinetic data in earlier in
clinical development, and to increase the efficiency of drug development. The
microdosing concept is based on using extremely low doses of a drug, which are
pharmacologically inactive but are able to delineate the pharmacokinetic profile
of the drug in humans [75, 76]. This strategy is also expected to reduce the
number of participants required for preclinical safety studies and to more
accurately predict the first-in-human doses.
The microdose of a small-molecule drug has been defined by the US and
European regulatory authorities as ââless than 1/100th of the dose calculated to
Anticancer Drug Development 69
yield a pharmacological effect of the test substance to a maximum dose of less
than 100 mg.ââ For a protein drug, 30 nmol is considered the maximum dose
[77â79]. One key consideration of microdosing studies is the requirement of
highly sensitive analytical methods. Such analytical methods include liquid chromatography
with tandem mass spectroscopy (LC/MS/MS), positron emission
tomography (PET), and accelerated mass spectroscopy (AMS). The use ofAMS,
however, requires the use of 14C radiolabeled drug, making it less popular.
The American College of Clinical Pharmacology recently issued a position
statement on the use of microdosing in the drug development process [80]. In this
chapter, Bertino et al. discussed the key considerations for the predictive success
and validation of utility of the microdosing protocol. They noted that the success
of microdosing strategy depends upon its ability to accurately predict the key
pharmacokinetic parameter estimates, e.g., bioavailability, clearance, and the
elimination rate, of a drug at much higher therapeutic doses of the drug. The
authors noted that only a few studies have reported the comparison of the
therapeutic with the microdose data. These studies, however, have used currently
marketed drugs and suffer from the limitation of âprior knowledgeâ, which helps
clinical study design in aspects such as the sampling intervals.
A significant limitation of microdosing studies is their inability to predict PK
parameters where drugs exhibit nonlinear pharmacokinetics. Nevertheless, this
new paradigm of anticancer drug development can complement the existing
animal-to-human dose-scaling strategies to improve the safety and the success
of early clinical trials.