Preclinical and clinical screening of drugs for anticancer activity (solid tumors)

Dr. Banhisikha Adhikari

OUTLINE

• Introduction• Preclinical screening In vitro methods In vivo methods

• Microdosing

• Newer Approaches

• Summary

• Clinical Screening Phase I Phase II Phase III Phase IV

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

Drugs widely used as cancer chemotherapeutic agent suffers from drawbacks like – High toxicity(bone marrow suppression, alopecia, nausea,

risk of secondary cancers) High cost Development of resistance Less tumor cell selectivity

Necessiates development of compounds with lesser toxicity, tumor cell selectivity, novel targets and more cost effective

For this quick and novel methods are being identified that can screen a large number of compounds

In vitro and in vivo models are systematically applied for screening of anticancer drugs

PRECLINICAL SCREENING

In Vitro methods In Vivo methods

In Vitro MethodsAdvantages• 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

• 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

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

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)×

100

Plates are read on an

ELISA reader at 570nm

Taken out of incubator

purple coloured formazan formed

thoroughly mixed with

isopropanol/DMSO

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

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

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

Examples of 3D modelsSpheroid 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

In Vivo methods• Aimed at predicting

Safe starting dose & dosage regimen for human clinical trialsThe 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

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

modelViral infection models:• Mouse mammary

tumor virus• Moloney murine

sarcoma virus• Newer genetically

engineered viruses

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

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 – cannot detect inhibition

of carcinogen activation

Other similar modelsCancer 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)

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.

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 – 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

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

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

Clinical screening

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

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

• 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

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.

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 Children’s Oncology Group Cancer and Leukemia Group

Trial featuresInclude 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

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

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)

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

Newer Approaches• Active immunotherapies: cancer vaccines stimulate a patient’s

immune system to destroy cancer cells— 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

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

Thank You