Evaluation of anticancer agents final

Evaluation of anticancer agents
- Dr Anup Thorat
- Guide : Dr Sandhya Kamat

History
5/4/2014 Evaluation of anticancer agents - Dr. Anup Thorat 2

History
Louise Goodman
Sidney Farber
Father of modern
chemotherapy
Alfred Gilman

Need for Novel Anticancer Agents
• Multidrug resistance.
• Long-term treatment with cancer drugs a/w 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 - more selective for tumour cells.

Pre clinical evaluation
IN VITRO IN VIVO

In vitro cytotoxicity studies:
• 1990 : NCI-60 screen
• Cytotoxicity assays on panel of human cancer cell lines
– MTT-assay
– SRB- assay
– 3H-thymidine uptake assay
– Fluorescence Dye exclusion tests
– Clonogenic assays
– Cell counting assay

In vitro cytotoxicity studies:
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 .

Cell lines
• Tumor cell lines derived from several cancer types
– lung, colon, melanoma, renal, ovarian, brain, and leukemia.
• The lines are prepared and cryopreserved using DMSO(di
methyl sulfoxide )
• THAWING OF THE CELLS : Rapid thawing of frozen ampoule in
water bath until it gets liquefied.

Cell lines
• The solution is centrifuged with saline for 10 mins to remove
the DMSO
• The saline is discarded and aliquot is taken for cell counting,
cell viability and for sub culturing.

CELL PROPERTIES & ASSAY
PROPERTIES ASSAY
ENZYMATIC PROPERTY MTT ASSAY
MEMBRANE INTEGRITY DYE EXCLUSION TEST
DNA CONTENT/REPLICATION STATUS 3H-THYMIDINE UPTAKE & FLUORESCENT ASSAY
PROTEIN CONTENT SRB ASSAY
COLONY FORMING POTENTIAL CLONOGENIC ASSAY
CELL DIVISION CELL COUNTING ASSAY

Microculture Tetrazolium Test(MTT Assay)
• A quantitative colorimetric assay
• Measures cellular growth, cell survival and cell proliferation
based on the ability of living cells.
MTT
(3-(4,5-
Dimethylthiazol-2-yl)-
2,5-
diphenyltetrazolium
bromide)
Formazan
mitochondrial dehydrogenase
in living cells only

MTT Assay - Procedure
• 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 inoculate in
multiwell plates.
• Cells are treated with various conc. of test compounds & the plate
incubated at 37 °C in 5% CO2 /95% humidified air (1-4 d)
• Cultures are taken out and 10 μl of MTT dye is added (5 mg/ml)
into each well and incubated for 4hrs

MTT Assay

MTT Assay - Procedure
• The plate is centrifuged, supernatant discarded & precipitated
formazan salt is dissolved in 100 μl of isopropanol/DMSO
• The plate samples are read at 570 nm microtiter plate reader
IC50 of drugs can be determined by counting the viable cells
% Cell viability = Absorbance of treated cells x 100
(%MTT reduction) Absorbance of untreated cells

Microtiter plate reader

SRB (SULPHORHADAMINE B) ASSAY
• Measures whole protein content which is proportional to the
cell number.
• SRB – a bright pink anionic protein staining dye that binds to
the basic amino acids of the cellular proteins.

SRB ASSAY - Procedure
• Cell lines are counted, cultured & inoculated in 96 well plates.
• After incubation with different concentrations of test
compounds, the cell cultures are stained with SRB dye.
• Washing with acetic acid removes the unbound dye and the
protein bounded dye is extracted using triss base
• Optical density is determined by 96-well plate reader

3H-Thymidine uptake assay
• Provides information about tumour kinetics, ploidy status of
the cells.
Procedure:
1. Tumor cell suspensions are exposed to the
drug for 5 days.
2. Radio-labeled 3H-thymidine is added
3. Replicating cells will incorporate 3H-thymidine into their DNA
4. Determined by autoradiography or liquid scintillation.

FLUORESCENCE ASSAY
• Cells are exposed to fluorescent labeled precursors after drug
exposure.
• Replicating cells will incorporate the dye into their DNA.
• Resulting fluorescence is measured by flow cytometry /
fluorescent microscopy .

Dye Exclusion Test
• This assay is relayed on the structural integrity of the cells.
• Dyes used : Trypan blue, Eosin, or Nigrosin
• Live cells possess intact cell membranes that exclude the dye,
whereas dead cells having lost membrane integrity take up the
dyes.

Dye Exclusion Test - Procedure
• Cells are incubated with different concentrations of test compounds for
4 days.
• Dead cells are stained with dye colour.
• Specimen is centrifuged & collected in microscopic slides.
• Live cells – stained with hematoxylin eosin.
• Tumor cell cytotoxicity is compared with control – duck erythrocytes.

Trypan blue dye exclusion test
Live
cell
Dead
cell

CLONOGENIC ASSAY
• Measures the growth inhibition.
• Important for the drugs that act by arresting the cells at checkpoints in the
cell cycle
Procedure:
1. Single cell suspensions are exposed to anticancer agents to be tested.
2. Suspensions are rinsed and plated in a semisolid medium.
3. After 14 to 28 days, some cells will having undergone several division
form tumour colonies which can be quantified in a visual or semi-
automated fashion.
4. No of colonies from treated cells is compared with untreated colonies.

CLONOGENIC ASSAY

Preclinical Toxicity Studies
• Aimed at predicting
(a) Safe starting dose & dosage regimen for human clinical trials(P1)
(b) The toxicities of the compound, &
(c) The likely severity and reversibility of drug toxicities.
• Regulatory requirement : Two acute preclinical toxicity studies
1. Rodent (mice) - single- and multiple-dose lethality studies.
2. Non rodent (dogs) - single- and multiple-dose confirmatory toxicity.
• Cytotoxic & non cyotoxic drugs

Acute Toxicity Studies
• First mouse given a single injection (IP, IV, SC, IM or PO) of 400 mg/kg (or
lower if the compound is extremely potent)
• Second mouse - 200 mg/kg &
• Third mouse - 100 mg/kg
• The mice are observed for a period of 2 weeks & sacrificed if there are
signs of significant toxicity
• If all 3 mice must be sacrificed, the next 3 dose levels (50, 35 and 12.5
mg/kg) are tested in a similar manner
• This process is repeated until a tolerated dose is found designated as MTD.

Preclinical Toxicity Studies
• To determine the phase I entry dose of a cytotoxic anticancer
agent, the dose levels that are lethal to 10, 50, and 90% of mice
(LD10, LD50, & LD90) is determined by the same route of
administration.
The projected phase I entry dose is usually 1/10th of the LD10
• A robust toxicity study needed to avoid lengthy phase I trials as
well as severe drug toxicities.

Why Mice?
• Mice are small, easy to handle.
• Short generation time & accelerated lifespan,manageable
costs, space, and time.
• Striking similarity to humans in anatomy, physiology, and
genetics.
• Over 95% of the mouse genome is similar to humans.
• Many of the genes responsible for complex diseases are shared
between mice and humans.
• Mouse genome can be directly manipulated.

Tumour Models
1. Carcinogen induced models
2. Viral infection models
3. Transplantation Models
– Murine
– Human
4. Genetically Engineered Mouse Models
5. In vivo hollow fibre assay

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 f/b 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

Mouse skin papillomas

MNU INDUCED RAT MAMMARY GLAND CA
• Induces hormone dependent tumors.
• Single i.v of 50 mg/kg of methylnitrosourea - 50 days old SD
rats.
• Adenocarcinoma will be produced within 180 days of post –
carcinogen – 75 to 95%
• Reduction in tumor size is compared
• Drawback – cannot detect inhibition of carcinogen activation.

RAT MAMMARY GLAND CA

DMBA induced rat mammary gland ca
• Sprague dawley rats
• Intragastric injection of 12 mg / kg of DMBA at 50 days of age
• Cancer appears within 120 days – 80 to 100%
• Detect the drugs inhibiting carcinogen activation.

MNU induced tracheal squamous cell ca
• Hamster
• 5% of MNU in normal saline is given once a week for 15 wks by
catheter.
• Within 6 months – 40 to 50%
• Test drug efficacy is measured by comparing with carcinogen
control group

DEN induced lung adenocarcinoma in Hamster
• 17.8mg DEN/kg body wt twice weekly by S.C inj for 20 weeks
starting at age 7 to 8 weeks.
• Usually produces tracheal tumors in 90-100% and lung tumors
in 40-50% of male syrian hamster.

Cancer site Cancer Type Species Carcinogen
Colon Adenocarcinomas Rat AOM (azoxymethane)
Prostate Adenocarcinomas Rat MNU (methylnitrosourea)
Esophagus Squamous cell
carcinoma
Rat NMBA(N-nitroso-
methylbenzylamine)
Breast Adenocarcinoma Mice NMU (N-Nitroso-N-
methylurea)

Viral infection 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 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.

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

Transplantation Models
• Disadvantages:
– Histopathology cannot be exactly correlated to human tumors
– Lack of step-wise progression through pre-neoplastic stages
– Evolution of tumor cells during passaging
– Requirement for angiogenesis to support the newly transplanted
tumor

Transplantation Models : Human Tumor Xenografts
• Athymic “nude”mice developed in 1960’s
• Mutation in nu gene on chromosome 11
• Phenotype: retarded growth, low fertility,
no fur, immunocompromised
– Lack thymus gland, T-cell immunity
• First human tumor xenograft of colon adenocarcinoma by Rygaard & Poulson,
1969

Xenograft Sites
• Subcutaneous tumor (NCI method of choice) with IP drug
administration
• Intraperitoneal
• Intracranial
• Intrasplenic
• Renal subcapsule
• Site-specific (orthotopic) organ inoculation

Xenograft Study Endpoints
• Toxicity Endpoints:
– Drug related death
– Net animal weight loss
• Efficacy Endpoints:
– Tumor weight change
– Treated/control survival ratio
– Tumor growth assay (corrected for tumor doubling time)

Xenograft Tumor Weight Change
• Tumor weight change ratio (used by the NCI in xenograft
evaluation)
• Defined as: treated/control x 100%
• Tumor weight in mg = (a x b2)/2
– a = tumor length
– b = tumor width
• T/C < 40-50% is considered significant

Human Tumor Xenografts
Advantages
• Many different human tumor cell
lines transplantable
• Wide representation of most
human solid tumors
• Good correlation with drug
regimens active in human lung,
colon, breast, and melanoma
cancers
Disadvantages
• Brain tumors difficult to model
• Different biological behavior,
metastases rare
– Survival not an ideal endpoint:
death from bulk of tumor, not
invasion
• Shorter doubling times than
original growth in human
• Difficult to maintain animals due
to infection risks

Transplantation Models : Cell line based allografts

Transplantation Models : Cell line based allografts
Advantages
• Very easy to maintain cell lines
indefinitely;
• Very fast to generate large
numbers of tumors
• Relative homogeneity of tumors
makes it easy to detect
differences
• Comparatively cheap
• Intact immune system
Disadvantages
• Mouse cells rather than human
• Inaccurate histopathology
compared to human tumors
• Evolution of tumor cells during
culture
• As it involves murine CA, ability
to predict response to therapy in
humans is controversial.

In Vivo Hollow Fibre Assay
• 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

Subcutaneous Hollow
Fibre implants

Clinical Evaluation
• The most reliable method for demonstrating efficacy is to
show a statistically significant improvement in a clinically
meaningful endpoint in blinded randomized controlled trials

Regulatory considerations
1. Regular Approval
– Longstanding route of drug approval based on the demonstration of
clinical benefit
2. Accelerated Approval
– Use of a surrogate endpoint that is reasonably likely to predict
benefit.
– Serious or life-threatening diseases
– Improvement over available therapy
– No existing therapy

Endpoints
• 1970s - Objective Response Rate (ORR)
• 1980s - improvement in survival, quality of life (QOL), physical
functioning, tumour related symptoms.
• Three kinds of end points have been used:
1. Overall Survival
2. Tumor assessment endpoints e.g. ORR,DFS, PFS,TTP, TTF; and
3. Symptom assessment endpoints e.g. palliation of side effects, QOL
scores.

Endpoint Regulatory
Evidence
Study Design Advantages Disadvantages
Overall
Survival
(OS)
Clinical benefit
for regular
approval
Randomized
studies essential
Blinding not
essential
1)Universally accepted
direct measure of
benefit
2)Easily & Precisely
measured
1)May involve larger studies
2)May be affected by crossover
therapy and sequential therapy
3)Includes non cancer deaths
Symptom
Endpoints
(patient
reported
Outcomes)
Clinical benefit
for regular
approval
Randomized
blinded studies
1)Patient perspective of
direct clinical benefit
1)Blinding is often difficult
2)Data are frequently missing or
incomplete
3)Clinical significance of small changes
is unknown
4)Multiple analyses
5)Lack of validated instruments
Objective
Response
Rate
(ORR)
Surrogate for
accelerated
Approval
or
regular
Single-arm or
randomized
studies can be
used
Blinding
1)Can be assessed in
single-arm studies
2)Assessed earlier and in
smaller studies
compared with survival
1)Not a direct measure of
benefit
2)Not a comprehensive
measure of drug activity
3)Only a subset of patients

Single-Arm Studies
• No available therapy
• ORR (objective response rate) and response duration in single-arm
studies can be a substantial evidence supporting accelerated
approval.
• Primary efficacy measure : Proportion of patients who achieve a
complete or partial response to the treatment.
• Design eliminates truly ineffective therapy

Single-Arm Studies
• Challenges :
1. Do not adequately characterize time-to-event endpoints (survival,
TTP, PFS)
2. Because of variability in the natural history of cancer, a randomized
study is necessary to evaluate time-to-event endpoints.
3. Inability to predict comparative performance vis-a`-vis the then-
available best possible, standard-of-care therapeutic option.

Non-inferiority Studies
• Rely on historical data to establish the active control’s treatment
effect size & constancy assumption
• Estimated size of the active-control’s treatment effect should be
based on a comprehensive meta-analysis of historical studies
• Challenges :
1. the estimation of active-control effect and the determination of amount
of effect (NI margin) to be retained. Usually large sample sizes.
2. Subsequent therapies and crossover to the active-control arm can
confound any NI analysis
3. NI trials with endpoints other than survival are problematic

Clinical Trial Phases
• What is the dose and schedule? Phase I
• Is it active?
– spectrum of anticancer activity ? Phase II
– is it better than standard therapy? Phase III
• Is it safe? Phase I to IV

Phase 0 : Microdosing
• The FIH dose : significant safety risk for the patient volunteers.
• Less than1/100th of the dose calculated to yield a pharmacological
effect of the test substance to a maximum dose of less than 100 µg.
• Advantages:
– Mitigate FIH dose risk,
– Gather early pharmacokinetic data (bioavailability, clearance, elimination
rate), and
– Increased efficiency of drug development.
• Highly sensitive analytical methods : PET, LCMS, accelerated mass
spectroscopy(AMS)

Phase 1
• Usually conducted in patients, rather than healthy volunteers.
• This adds to the challenges :
1. Recruitment of tumor-specific patient volunteers
2. The recruited volunteers may be in the advanced stages of the
disease - refractory to the currently available standard-of-care
treatment options - cost escalation.

Dose Escalation Studies
• Phase 1 entry dose is obtained from preclinical toxicity studies
• Endpoints :
– Along with MTD, info on clinical toxicity, pharmacokinetics, and
preliminary antitumor activity is obtained.
– Also for cytotoxic agents, dose-related toxicity is regarded as a
surrogate for efficacy.
Progressively escalating
doses
MTD

Dose Escalation Studies
• Modified Fibonacci search :
1. Safe starting dose(d) = 1/10th of the LD10
2. Enroll patients in cohorts of three, and escalate the dose according
to a modified Fibonacci sequence
3. Higher escalation steps have decreasing relative increments -100,
65, 52, 40, 29% & thereafter 33% increases over the previous dose.
4. Ethical issues : substantial numbers of patients are treated at
nontherapeutic doses
5. Efficiency issues : Lengthy trials, time & cost incurred.

Dose escalation : Other Methods
• Pharmacologically guided dose escalation (PGDE)
– Using the preclinical toxicology data to rapidly escalate doses to a
target area under the curve (AUC) value obtained from murine
pharmacokinetic data.
• Non-pharmacokinetic statistical modeling approaches :
– Statistical approaches model the dose–toxicity relationship as a
sigmoidal curve to predict the MTD. The toxicity is then evaluated &
actual MTD found by rapid dose titration.

Ethical Challenges In Phase I
1. The paucity of benefits with substantial risks
2. Informed consent
• Majority of participants are treated at doses that cannot produce
responses in human tumors.
• The objections based on informed consent are deficiencies of
disclosure, understanding, and voluntariness
• Vulnerable group – Terminally ill patient

Phase II and III Clinical Trials
• Phase II studies are carried out in a small group of patients with a
specific tumor type to
– Determine anticancer efficacy &
– To define the therapeutic window of the compound
• Any arm may be terminated early because of discouraging results,
and the response rate of each arm is assessed separately against a
historical control rate with definable α and β error probabilities
• Phase III trials are conducted in a much greater number of patient
volunteers of the selected tumor type with prospective and
randomized evaluation against standard of care.

• Embedding a randomized phase II study within a phase III
study
– Patients who progress on one of the arms of Phase II would then be
randomly assigned to arms E or C of phase III & and
– Their survival data would be combined in a stratified fashion with
data from patients directly assigned to arms C or E for the phase III
assessment.
– This design allows untreated patients to be enrolled onto the phase II
study, while assuring them a more established therapy if they
progress

• Phase II studies act as a screen of antitumor efficacy to select
the most promising agents to enter the pivotal phase III clinical
trials.
• A Phase II study should efficiently eliminate truly ineffective
therapy & reliably indicate whether subsequent phase III
testing is warranted.

Evaluation of anticancer agents final

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