Eval of anticancer:

Need for new drugs

  • Despite the major advances that have been made in the treatment of malignancy, many patients still die either from unresponsive tumors or recurrent disease
  • Need for anticancer drugs that would eradicate cancer cells without harming normal tissues
  • Need for anticancer agents that would target resistant cancer cells
  • Human tumor stem cell (HTSC) assay/ Clonogenic Assay
  • Disease-orientated – model system incorporating multiple tumors cell of the same histotype
  • Soft agar colony growth of freshly explanted human tissue is taken
  • Outcomes are based on growth inhibition
  • Initial studies found that most drugs were active with the exception of drugs requiring systemic activation
  • HTSC assay – for evaluating efficacy of clinically active agents and individualizing cancer treatment


  • Low plating efficiency of most solid tumors
  • There are no phase III clinical studies of individualized therapy demonstrating a significant increase in survival compared with empirically determined standard treatment
  •  Therefore, the HTSC assay has not found a role in the individualization of patient therapy
  • Human tumor cell line assay
  • The initial cell line panel incorporated a total of 60 different human tumor cell lines of diverse histology
  • Different types of cancer including drug-resistant cell lines included
  • nonclonogenic protein stain sulfo-rhodamine B assay (38) is used to determine cell viability
  • Each compound is tested over a 5-log concentration range against each of the 60 cell lines
  • These data generate characteristic profile or ‘‘fingerprint’’ of cellular response, i.e.  the ‘‘mean graph’’
  • ‘‘COMPARE’’ is a computerized, pattern recognition algorithm used in evaluating and exploiting the fingerprint data in order to determine the degree of similarity between mean graph profiles generated by similar or different compounds


  • The sensitivity of a cell line, along with knowledge of its molecular characteristics may indicate that a compound’s action is mediated by its interaction with molecular target
  • Alternatively, differential expression in the form of a mean graph may indicate why particular cell lines may be resistant to a test compound
  • Endpoints –
  •  GI50 (concentration required to inhibit 50% of cells)
  •  Total growth inhibition
  •  LC50 (concentration  required to kill 50% of cells)


  • Factors other than the inherent chemo sensitivity of tumor cells significantly influence the outcome of chemotherapy in vivo (e.g., pharmacokinetics, tumor micro regions/pH)
  • Not  sufficiently discriminatory to ensure that only a relatively small number of compounds would be selected for further evaluation in human tumor xenograft models

Preclinical toxicity study

  • Aimed at predicting
    • Safe starting dose & dosage regimen for human clinical trials(P1)
    • The toxicities of the compound, &
    • The likely severity and reversibility of drug toxicities.
  • Regulatory requirement : Two acute preclinical toxicity studies
    • Rodent (mice) – single- and multiple-dose lethality studies.
    • 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 receives a dose of 200 mg/kg and a third mouse receives a single dose of 100 mg/kg
  • The mice are observed for a period of 2 weeks
  • They are sacrificed if there are signs of significant toxicity
  • If all 3 mice must be sacrificed, the next 3 dose levels (50, 25 and 12.5 mg/kg) are tested in a similar manner This process is repeated until a tolerated dose is found
  • This dose is then designated as MTD
  • For the standard hollow fiber assay (HFA), the high and low dose levels are determined using the MTD the formula below
  • High dose = [MTD x 1.5]/4
  • Low dose = 0.67 x high dose

Hollow fiber assay

  • Mice treated with experimental agents starting on day 4 following fiber implantation and continuing 4 days
  • Each agent  administered by i.p injection at 2 dose levels. The doses are based on the maximum tolerated dose (MTD)
  • The fibers are collected from the mice and subjected to the MTT assay
  • The percent net growth in each treatment group is calculated and compared to the percent net growth in controls
  • A 50% or greater reduction in percent net growth in the treated samples compared to the vehicle control samples is considered a positive result


  • The HFA assesses the pharmacologic capacity of compounds to reach physiologic compartments and shows a practical means of quantifying viable tumor cell mass
  • It is relatively rapid and cost-effective demonstration of in vivo activity
  • HFA as an in vivo prescreen is good predictor of in vivo xenograft activity which is the next step in preclinical screening

Human tumor xenograft models

  • Xenograft tumors are established by the s.c inoculation of tumor cells into nude mice
  • Growth of solid tumors monitored using in situ caliper measurements
  • Activity is defined by
    • Tumor growth delay
    • Net tumor cell kill T/C ( T/C – median treated tumor mass/median control tumor mass)
  • Drug-related deaths and body weight loss are used as parameters of toxicity


  • Requirement for an immunocompromised host
  • Not all tumor systems can be studied by xenograft model
  • Inability of these models to fully resemble the complex relationship between the tumor and its microenvironment (e.g., angiogenesis)
  • Most importantly, the ability of xenografts to predict drug efficacy in human cancer patients has been disappointing

Orthotopic and metastatic tumor models

  • Organ environment can influence the response of tumors to chemotherapy
  • As s.c. tumor models are not representative of the primary tumor site
  •  In addition, clinically we treat well-established and advanced metastatic disease
  • Thus, orthotopic tumor models seem to be more representative of a primary tumor with respect to tumor site and metastasis
  • Advantage – orthotopic model is targeting processes involved in local invasion (eg, angiogenesis) & is undertaken at a more clinically relevant site
  • Disadvantage –
  1. need for a high level of technical skill, time, and cost
  2. Therapeutic efficacy more difficult to assess with orthotopic models in contrast to the relative ease of s.c. tumor 

Autochthonous models

  • Autochthonous tumors include spontaneously occurring tumors and chemical, viral, or physical carcinogen-induced tumors
  • Advantages of autochthonous tumors include orthotopic growth, tumor histology devoid of transplantation introduced changes, and metastasis via lymphatic and vascular vessels
  • Outcomes compared from autochthonous models and GEMs found that autochthonous models correlated best with clinical responses


  • Autochthonous tumor models have an inherent variability in the time, frequency, number of tumor(s) induced and thus the number of animals required
  • Time required – several months to a year, as opposed to weeks with transplanted xenograft models
  • Thus, autochthonous tumor models are best reserved for confirmation

Genetically engineered mouse models

  • Genetically engineered mice are predisposed to cancer by introducing cellular/viral oncogenes into the mouse germ line
  • Have highlighted the importance of specific oncogenes and tumor suppressor genes
  • GEM models resemble genetic/molecular changes in human cancer & are used to test novel anticancer therapeutics
  • Genes targeted to mouse embryonic stem cells, result in oncogene -bearing transgenic mice i.e., gene knockout mice


  • GEM models possess well validated drug targets and may potentially offer a more appropriate preclinical model
  • Additionally, GEM tumors develop autochthonously /in situ and therefore may be more biologically representative of a particular tumor type in humans


  • GEM models are expensive and time consuming
  • Their use is often restricted by intellectual property rights and patents
  • In addition to embryonic lethality, mice often do not develop the expected tumor type as they may die prematurely
  • The genetic background can affect transgene expression where outcome like rapidity of lesion ,type of lesion and tumor histiotype are affected by genetic background
  • Clinical evaluation general points
  • Single arm studies
  • Non inferiority studies
  • Design for protectants
  • Endpoints
  • DFS (Disease free survival): time from randomization to tumor recurrence or death d/t any cause
    • Used in the setting of-
    • After definitive t/t
    • When a large percentage of patients achieve complete responses with chemotherapy
    • In situations where survival may be prolonged, making a survival endpoint impractical
  • ORR (Objective response rate): proportion of patients with a tumor size reduction to a predefined amount
    • ORR is a direct measure of drug antitumor activity, which can be evaluated in a single-arm study
  • TTP (Time to progression): time from randomization until predefined tumor progression. Doesn’t include death
  • PFS (Progression free survival): TTP and death
  • Whether an improvement in PFS represents a surrogate for clinical benefit depends on the magnitude of the effect & risk-benefit compared to available therapies
  • Define tumor progression criteria in the protocol
  • Visits and radiological assessments should be symmetric between the two study arms to prevent systematic bias
  • Studies should be blinded
  • TTF (Time to treatment failure): time from randomization to t/t discontinuation d/t any cause [disease progression, toxicity or death]. But it cannot be a regulatory endpoint since it doesn’t distinguish efficacy from other parameters
  • Biomarkers
  • Generally, biomarkers assayed from blood or body fluids have not served as primary endpoints for cancer drug approval
  • Further research is needed to establish the validity of available biomarkers and determine whether improvements in biomarkers predict clinical benefit
  • Biomarkers can be useful in identifying prognostic factors and stratification factors to be considered in study designs.
  • Phases
  • Phase 1:
  • Design: Conventional Fibonacci approach OR Statistically based dose escalation model.
  • Ethical issues.
  • Endpoints. Response rate, toxicity and clinically meaningful endpoints
  • Phase 2:
  • Randomized selection design
  • Design with reference standard t/t control arm
  • Phase 2/3 study design

Leave a Reply

Your email address will not be published. Required fields are marked *

This site uses Akismet to reduce spam. Learn how your comment data is processed.