Transcript CLASSIFICATION OF CYTOTOXIC AGENTS

Pharmacologic Anti-Cancer Treatments Seminars 2007:
Part 1:
Tumor Biology and Kinetics
Introduction of Cytotoxic Agents
Carlos Linn, M.D.
林錦洲 醫師
Clinical Research Physician, Oncology
Lilly Oncology
Board Certified Gynecologic Oncologist
Cellular Kinetics
•
Human body contains 5x1013 cells
•
Cells can either be
- non dividing and terminally differentiated
- continually proliferating
- rest but may be recruited into cell cycle
•
Tumour becomes clinically detectable when there is a
mass of 109 cells (1g)
2
The Cell Cycle
DEATH
G0
DIFFERENTIATION
Mitosis
M
DNA content = 4n
G2
G1
DNA content = 2n
S
DNA synthesis
3
The Cell Cycle
4
Cancer Cells and Normal Cells
CANCER CELLS
NORMAL CELLS
Frequent
mitoses
Normal
cell
Nucleus
Blood vessel
Few
mitoses
Abnormal
heterogeneous cells
Loss of contact inhibition
Oncogene expression is rare
Increase in growth factor secretion
Intermittent or coordinated
growth factor secretion
Increase in oncogene expression
Loss of tumor suppressor genes
Presence of tumor suppressor
genes
5
Growth Factors and Oncogenes
Paracrine (Adjacent cells)
Growth Factor
and Receptor
Synthesis
Growth
Factor
Growth Factor
Receptor
Post
receptor signal
transduction
pathways
Gene Activation
Oncogenes
6
Oncogenesis
NORMAL GROWTH AND
DEVELOPMENT
NORMAL EXPRESSION &
RESPONSIVE ONCO SUPPRESSION GENE
CELLULAR
ONCOGENE
MUTAGENIC or
CARCINOGENIC AGENTS
VIRAL ONCOGENE
INCREASED OR ABNORMAL
EXPRESSION
CANCER
GROWTH
7
Example:
Oncogenesis Integrated by HPV
Integration of
HPV DNA
genome E6, E7
into Host-cell
Immortally
malignant
NO more
Koliocytosis
 Virus stops
duplication
Complete viral
life cycle with
Koliocytosis
 Virus
duplication
Beutner, KR et al, "Human Papillomavirus and Human Disease." Am J Med 1997; 102(5A):9-15.
8
E6, E7 Protein involvement in cell cycle
regulation
Cell cycle proteins, influenced by
E6, E7 proteins
E6 Bind and Degrade p53:
Loss of p53-induced
apoptosis/G1 arrest of the
cell cycle; reduces p53
protein via degradation.
E7 releases the E2F transcription
factor by binding Rb
(retinoblastoma protein),
promoting cell cycle
progression
transcriptional deregulation of cell
cycle control, uncontrolled cell
proliferation
intracellular control - cyclindependent kinase inhibitors (CKI)
9
CYCLIN DEPENDENT KINASES
tyr15-P
thr14-P
P-thr161
- protein kinase
- binds to cyclin
- kinase domain
- regulatory domain
- present throughout cell cycle
e.g. cdk1 (= cdc2)
10
CYCLINS
- No intrinsic enzymatic activity
- Binds cdk
- Synthesized and degraded each cycle
- Essential component for cdk activity
e.g. Cyclin B
11
CYCLIN / CDK
• Regulated by:
•
•
- tyr15 phosphorylation
P-thr161
• inhibitory kinases
• activating phosphatases
- Direct interaction
• inhibitory proteins
• p21, p27, p57
• p16, p15, p18,p19
tyr15-P
thr14-P
cdk1
(cdc2)
cyclin B
12
CELL CYCLE CHECKPOINTS
CYCLIN A / cdk 2
S
G2
CYCLIN B / cdk 1
CYCLIN E / cdk 2
G1
M
CYCLIN D / cdk 4,5,6
13
Variation in Cell Cycle Cyclins
Cyclin-dependent kinases (CDK)
CDK 4
CDK 2
CDK 1
Cyclins
M
D
G1
E
A
S
B(A)
G2
M G1
Start
Cell cycle phases
14
Cell Cycle
RNA, Protein
Mitosis, Cytokinesis
Lamin
H1
Abl
Cyclin B/A
CDK1
DNA, RNA,
Protein
G2 M
3-4 h 1 h
S
6-8 h
Cyclin D’s G0
CDK4,6
G1
6-12 h
RNA, Protein
Cyclin A
Cyclin E
CDK2
CDK2
p53
pRb
15
E6, E7 involvement in cell cycle regulation
DNA damage
Phosphorylation
16
DNA Damage - Cell Cycle Arrest
Damage Dependent Checkpoints
G1 - S - G2
G1 - S - G2
CELL
No.
wild-type
DNA content
Asynchronous
DNA content
X-ray treated
G1/S block
G2/M block
(6-9 hours)
Loss of G1/S in
p53 deficient
cells
17
G1/S CHECKPOINT
IN RESPONSE TO DAMAGE
X-rays
P-tyr15
strand
break
cdk2
ATM
p53
p21
cyclin E
p21 = CKI class (cyclin dependent kinase inhibitors)
N-terminal of p21 forms complex with cyclin / cdk inhibit kinase
18
Cell Cycle Regulation
1. CDK phosphorylation
DNA damage
2. C degradation
Active p53
3. C & CDK synthesis
4. CDK inhibition
CDK2
CE
p21
pRb
P
pRb
E2F
Enzymes for
DNA synthesis
Passage from
G1 to S
19
Growth Factors & Cell Cycle
Gene Transcription
+
Receptors
S
Priming
G0
G1
Cell Cycle
G2
M
20
Retinoblastoma protein (pRb) & CDK
inhibitors: p21, p27, p16
21
The Normal Cell Cycle &
“Cyclins” of the cell cycle
E6, E7: immortalize
human keratinocyte
E5 protein
G1 arrest
Normal cell cycle (with tumor
suppression and apoptosis)
Neoplastic cells
(immortal)
22
Common Chemotherapeutic Agents
•
Alkylating agents
•
Antimetabolites
•
Antitumor Antibiotics
•
Alkaloids
•
Taxanes
23
Classes of antineoplastic drugs
•
•
•
•
•
•
•
•
•
•
•
Alkylating agents
Interact directly with cellular DNA
Antimetabolites
Resemble cellular metabolites (folic acid, purine, pyrimidine)
Interfere with DNA precursors & cellular metabolism
Antitumor antibiotics
Derived from soil fungus, some antiinfective activity
Interfere with DNA activity
Mitotic Inhibitors
Derived from plant extracts
Interfere with formation of mitotic spindle, arresting mitosis
24
Antineoplastic Agents
Alkylating agents
Carboplatin, cyclophosmamide, melphalan,
thiotepa
(Form bonds with nucelic acids and proteins)
Antimetabolites
Methotrexate, fluorouracil, gemcitabine
(similar to metabolites involved in nucelic acid synthesis)
Natural Products
doxorubicin, docetaxel, vinolbine, topotecan
(anti tumour antibiotics,mictotubule stabilizer, mitotic inhibitor, topoisomerase inhibiotor)
Endocrine agents
Anastrozole, tamoxifen, prednisolone, goserelin
(Aromatase inhibitors, oestrogen antagonist, corticosteroids, LHRH agonist)
Molecularly targeted agents
Retinoids, trastuzumab, gefitinib
(gene expression, monoclonal antibody, tyrosine kinase inhibitor)
Biologic response modifiers
Interferon, thalidomide, filgrastim
25
Alkylating Agents
•
Interact with DNA causing substitution reactions,
cross-linking reactions or strand breaks
•
Example: cisplatin
26
Antimetabolites
•
Cytotoxic effects via similarity in structure or
function to naturally occurring metabolites involved
in nucleic acid synthesis—either inhibit enzymes
involved in nucleic acid synthesis or produce
incorrect codes
•
Example: methotrexate, pemetrexed, gemcitabine,
5-FU
27
Antitumor Antibiotics
•
Group of related antimicrobial compounds produced
by Streptomyces species in culture
•
Affect structure and function of nucleic acids by:
– Intercalation between base pairs (doxorubicin),
– DNA strand fragmentation (bleomycin),
– Cross-linking DNA (mitomycin)
28
Alkaloids
•
Bind free tubulin dimers
•
Disrupting balance between microtubule
polymerization and depolymerization
•
Arrest of cells in metaphase
•
Examples: vincristine, vinblastine, vinorelbine
29
Taxanes
•
Disrupt equilibrium between free tubulin and
microtubules
•
Stabilization of cytoplasmic microtubules
•
Formation of abnormal bundles of microtubules
•
Examples: paclitaxel and docetaxel
30
Paclitaxel & Docetaxel
1971
Pacific Yew: Taxus brevifolia
OH
1986
European Yew: Taxus baccata
31
Classification of Cytotoxic Agents
ALKYLATING
AGENTS
ANTIMETABOLITES
MITOTIC
INHIBITORS
ANTIBIOTICS
OTHERS
BUSULFAN
CYTOSINE
ETOPOSIDE
BLEOMYCIN
L-ASPARAGINASE
CARMUSTINE
ARABINOSIDE
TENIPOSIDE
DACTINOMYCIN
HYDROXYUREA
CHLORAMBUCIL
FLOXURIDINE
VINBLASTINE
DAUNORUBICIN
PROCARBAZINE
CISPLATIN
FLUOROURACIL
VINCRISTINE
DOXORUBICIN
CYCLOPHOSPHAMIDE
MERCAPTOPURINE
VINDESINE
MITOMYCIN-C
IFOSFAMIDE
METHOTREXATE
TAXOIDS
MITOXANTRONE
MELPHALAN
GEMCITABINE
TAXANES
PLICAMYCIN
PEMETREXED
ANTHRACYCLINES
EPOTHILONES
32
Sites of Action of Cytotoxic Agents –
Cell Cycle Level
Antibiotics
Antimetabolites
S
(2-6h)
G2
(2-32h)
M
(0.5-2h)
Vinca alkaloids
Mitotic inhibitors
Taxoids
Alkylating agents
G1
(2-h)
G0
33
Types of chemotherapy
• Cell cycle dependent
– Cell cycle phase specific
• Cell cycle independent
– Cell cycle phase non-specific
34
Cycle-Specific Agents
35
Sites of Action of Cytotoxic Agents –
Cellular Level
DNA synthesis
Antimetabolites
DNA
DNA transcription
Alkylating agents
DNA duplication
Intercalating agents
Mitosis
Spindle poisons &
Microtuble Stablizers
36
Sites of Action of Cytotoxic Agents
PURINE SYNTHESIS
6-MERCAPTOPURINE
6-THIOGUANINE
PYRIMIDINE SYNTHESIS
RIBONUCLEOTIDES
METHOTREXATE
5-FLUOROURACIL
HYDROXYUREA
PEMETREXED
DEOXYRIBONUCLEOTIDES
ALKYLATING AGENTS
AKYLATING LIKE
CYTARABINE
GEMCITABINE
(INTERCALATING)
ANTIBIOTICS
DNA
ETOPOSIDE
RNA
TOPOISOMER
PROTEINS
L-ASPARAGINASE
VINCA ALKALOIDS
ENZYMES
MICROTUBULES
TAXOIDS
37
Drug Resistance
EXTRACELLULAR
PGP170
INTRACELLULAR
ATP
Drug
ATP
Drug
Plasma
Membrane
38
Mechanisms of Taxane Resistance
Altered metabolism
by host
Effect of tumor
growth kinetics
Taxanes
P-gp mediated
drug efflux
Tubulin binding site
mutations
Inhibition of apoptotic
signaling
P-gp = P-glycoprotein.
Dumontet and Sikic. J Clin Oncol. 1999;17:1061.
39
Taxane Resistance Mediated through
Multidrug Resistance (MDR)
MDR is mediated by mdr1
gene amplification
encoding P-gp
Extracellular
•
P-gp is a cell membrane
protein
1
•
Overexpressed in some
chemoresistant tumors
•
In chemosensitive tumours,
can be upregulated after
therapy
•
Anthracyclines, taxanes,
vinca alkaloids are P-gp
substrates
2
3
4
5
6
NBF1
NH2
7
8
membrane
•
9 10 11 12
NBF2
COOH
Intracellular
NBF = nucleotide binding factor
40
Anti-Folate Transporters
Reduced Folate Carrier (RFC)
THFs
Methotrexate, 5-FU,
Raltitrexed (Tomudex)
Pemetrexed (ALIMTA®)
Folate Receptor (FR-α)
Rothberg KG et al., J Cell Biol. 110: 637-649, 1990.
Folic Acid, THFs
CB 3717l
Pemetrexed (ALIMTA®)
Efflux by MRP
Westerhof GR et al., Mol. Pharmacol 48: 459-471, 1995
Zhao R et al., Clin Cancer Res 6: 3687-3695, 2000
Pratt SE et al., Proc. Am. Assoc. Cancer Res 43: 782, 2002
Methotrexate
Pemetrexed
(ALIMTA®)
41
Multiple Drug Resistance Proteins &
Anti-Folate Drug Resistance
Reduced Folate Carrier
Anti-folate
Anti-folate
RFC
Low affinity for folic acid
High affinity for
antifolates
High activity in
malignant tissue
ALIMTA
Folate
receptor
Membrane Folate Receptor
Anti-folate MFR
Anti-folate
ADP
High affinity for folic acid
Low affinity for antifolates
High expression in certain malignancies
(mesothelioma, ovary)
MRPs
ATP
(cell membrane)
MDRs: Multiple Drug
Resistance Proteins
42
Tumour kinetic
Growth rate depends on:
growth fraction
-percent of proliferating cells within a given system
-human malignacy ranges from 20-70%
-bone marrow 30 %
cell cycle time
-time required for tumour to double in size
rate of cell loss
43
Doubling times of some human
tumours
Tumour
Doubling times (days)
Burkitt’s lymphoma
1.0
Choriocarcinoma
1.5
Hodgkin’s disease
3-4
Testicular embryonal carcinoma 5-6
Colon
80
Lung
90
44
Tumor Kinetics – Original Hypothesis
•
Conventional views in the field of oncology support the
notion that:
– tumor growth is exponential
– chemotherapy treatment is designed to kill in log
intervals (kills constant fractions of tumor)
•
Currently, chemotherapy for ovarian cancer is administered
in 3-week intervals.
•
Combination therapy and increased drug dose levels aim at
improving ovarian cancer chemotherapy.
45
Gompertzian Growth
•
Growth rates are exponential at early stages of
development and slower at later stages of development.
- Biological growth follows this characteristic curve.
46
Gompertzian growth model
Initial tumour growth is first order, with later growth
being much slower
Smaller tumour grows slowly but large % of cell
dividing
Medium size tumour grows more quickly but with
smaller growth fraction
Large tumour has small growth rate and growth
fraction
47
Tumor Growth
number of
cancer cells
10 12
diagnostic
threshold
(1cm)
10 9
time
undetectable
cancer
detectable
cancer
limit of
clinical
detection
host
death
48
Rationales in Human Cancers
•
Small tumors grow faster than larger tumors
•
Human cancers grow by non-exponential
Gompertzian kinetics
49
Principle of chemotherapy
First order cell kill theory
- a given dose of drug kills a constant
percentage of tumour cells rather than an
absolute number
Maximum kill
Broad coverage of cell resistance
50
Theoretical Tumor Kinetics
Tumour
Surviving cells
tumour kill (%)
untreated
90 (1-log)
99 (2-log)
99.9 (3-log)
99.99 (4-log)
109
108
107
106
105
Viable mass
1g
100mg
10mg
1mg
100μg
Recovery of
(doubling time)
3.33 days
6.66 days
9.99 days
13.3 days
51
3 LOG KILL, 1 LOG REGROWTH
TUMOR CELL NUMBER
Chemotherapy
Time
52
Hypothesis of Alternative Intervals
The rate of tumor volume regression is proportional to the
rate of growth.
Tumors given less
time to grow in
between treatments
are more likely to
be destroyed.
Tumor cell regrowth can be prevented if tumor cells
are eradicated using a denser dose rate of cytotoxic therapy.
53
Principle of chemotherapy
Rationale for combination chemotherapy
Different drugs exert their effect through different
mechanisms and at different stages of the cell cycle,
thus maximize cell kill
Decease the chance of drug resistance
54
Thanks for Your Attention
To Be Continued…..
55
Example:
Metabolism of Cyclophosphamide
CYCLOPHOSPHAMIDE
HEPATIC
CYTOCHROMES
P 450
ACTIVATION
INACTIVATION
4-KETOCYCLOPHOSPHAMIDE
CARBOXYPHOSPHAMIDE
ALDEHYDE
4-OH CYCLOPHOSPHAMIDE
DEHYDROGENASE
ALDOPHOSPHAMIDE
ACROLEIN
PHOSPHORAMIDE
MUSTARD
TOXICITY
CYTOTOXICITY
56