Chemotherapeutic Agents -Introduction

Alkylating agent Platium Antimetabolite Topoisomerase poison Antimicrotubule Others

Cell Cycle damage: DNA

DNA duplication

Radiation, Platiums alkylating agent topoisomerase inhibitors Anti-metabolites

Protein G1 synthesis Mitosis

G2

Protein synthesis

G0

Anti-microtubules

Antimetabolite
 Folate
  

analog

Methotrexate Pemetrexed 5-FU(IV and oral)

 Purine/Purimidine
  

analog

C: Cytarabine(ara-C), Gemcitabine Cytarabine(araA: Fludarabine U: 5-FU 5-

5-FU (Fluorouracil)
 

Developed by Heidelberger and patented in 1957 remains at the very core of most chemotherapeutic approaches to colorectal cancer effectively metabolized by the same enzymatic pathways as uracil Leucovorin
PPO 7th edition

bolus

CIF
5-formyl-tetrahydrofolate

DPD: dihydropyrimidine dehydrogenase TS: thymidylate synthase

MTX

Clinical Colorectal Cancer 2002;1(4):220-9

Oral 5-FU 5UFUR

Capecitabine
The Oncologist 2002;7:288-323

Cytarabine (Ara-C) (Ara Ara-C Ara 

Cytidine deaminase DCK AraAra-CMP

Ara-U
AraAra-CTP

dCMPdCMP-K AraAra-CDP NDPNDP-K dCMP deaminase AraAra-UMP

Mechanism: inhibition of DNA synthesis Most sensitive in S phase Cross BBB in high dose (7-14% of serum level) Clinical: solid & hematological malignancy Toxicity: myelosuppression, cerebellar, N/V Prophylactic steroid for rash and conjunctivitis
PPO 7th edition

Gemcitabine (Gemzar)
 dFdC

Gemzar (dFdC)

dCK dFdCMP d FdCTP=>DNA termination  Toxicity: BM, transient fluflu-like in 45% patients, patients, asthenia, liver, lung, HUS  Clinical: NSCLC, pancreatic, bladder cancer
PPO 7th edition

CamptothecinCamptothecin-Mechanism

PPO 7th edition

TopoTopo-II Function

PPO 7th edition

Anti-HIV Agents Non-Symmetrical Cyclic Ureas

S N O N NH O N S HN N O

P2 SAB AA
R4
N

P2'
O
N

R3 R2

SAB ALD P1'

R1

HO

OH

P1

HO

OH

A. XV-638

B. cyclic urea ring

850(CA1) x 735(ALD2) x 1350(SAB3) x 1350(SAB4) ~ 1.1 x 1012 cyclic ureas fragment based focusing + analog based focusing + structure based focusing 5(CA1)
x

5(ALD2)

6(SAB3)

6(SAB4) = 900 cyclic ureas

HO OH O N O HO N OH O O R3

R4

R1
Analog 5-3-16-10 positioned at the catalytic site HIV PR

R2

Analog 1-2-8-9

V. Frecer, E. Burello, S. Miertus, Bioorg. Med. Chem. (2005)

METHYLATION
 protects

DNA from mutation  depends upon methionine (SAM), folic acid, vitamin B12  enhanced by dimethylglycine (DMG), choline, betaine  CAVEAT: methylation inactivates genes; aberrant methylation may inactivate tumor suppressor genes

Pathophysiology and Pharmacology of Reactive Oxygen V. Bauer, . Mtys, S. tolc, R. Species (ROS) Sotnkov,
V. Nos ov
Reactive Oxygen Species as Mediators of Tissue Injury, Diseases and their Pharmacology

Institute of Experimental Pharmacology, Slovak Academy of Sciences, Bratislava, SK

The beginnings

1775 - Priestley:
discovery of O2 observation of toxic effect of O2

1900 - Gomberg:
discovery of triphenylmethyl radical

Until 1950/60:
minimal attention was given to biological actions of free radicals and reactive oxygen species

Evidence on the existence of ROS 1954 - Gerschman et al. : Recognition of

similarities between radiation and oxygen toxicity

1969 - McKord and Fridovich:

Discovery of superoxide dismutase and suggestion of the existence of endogenous superoxide 1973 - Babior et al.: Recognition of the relationship between superoxide production and bactericidal activity of neutrophils 1981 - Granger et al.: recognition of the relationship between local ROS production and ischemia/reperfusion induced gut injury

Free radicals have one or more unpaired electrons in their outer orbital, indicated in formulas as [y]. As a consequence they have an increased reactivity with other molecules. This reactivity is determined by the ease with which a species can accept or donate electrons. The prevalence of oxygen in biological systems means that oxygen centered radicals are the most common type found. O2 acts in a process that is central to metabolism in aerobic life, as a terminal electron acceptor, being reduced to water. Transfer of electron to oxygen

The term reactive oxygen species (ROS) rather than oxygen radicals is now generally preferred because singlet oxygen (its one form), hydrogen peroxide, hypochlorous acid, peroxide, hydroperoxide and epoxide metabolites of endogenous lipids and xenobiotics have chemically reactive oxygen containing functional groups, but are not radicals and do not necessarily interact with biological tissues via radical reactions. Molecular oxygen is a biradical, having two unpaired electrons of parallel spin. As it is a terminal electron acceptor being reduced to water, oxygen acts in

Reactive Oxygen Metabolite Cascade (ROM Cycle)

4O2 XO
at presence of NADPHO

Cytochrome Cascade
4O2 4e4O24e4O224e-

4e4O2.-

SOD 4O234e2H2O2 + 2O2 4O2416H+ 8H2O

4O2 + 8H2O 4H+

CAT

2H2-O + + 4e 2H2O + 3O2 O2

4O2 + 16H+ + 16e(yields to production of

Half-life of some reactive Halfreactive species

Reactive species HalfHalf-life (s) 10-9 Hydroxyl radical (yOH) 10-6 Alcoxyl radical (ROy) 10-5 Singlet oxygen (1O2) 0.05 1.0 Peroxynitrite anion (ONOO-) 7 Peroxyl radical (ROOy) 1 - 10 Nitric oxide (yNO) minutes/hours Semiquinone radical pontan. hours/days Hydrogen peroxide (H2O2) spontan. hours/days
(accelerated by enzymes) enzymes

Physiol conc. (mol/l) mol/l)

10-9 10-9 - 10-7 10-12 - 10-11

Superoxide anion (O2y-)

spontan. hours/days pontan. hours/days

(by SOD accel. to 10-6)

Hypochlorous acid (HOCl) dep. on substrate Hypochlorous

ROS present in mammalian tissues have both endogenous and exogenous origin. Their production is essential to normal function or metabolism of most mammalian cells. Approximately, 90% of all oxygen consumed by mammalian cells is catalytically reduced by four electrons to yield two molecules of water. It is now clear that oxygen may also be reduced by less than four electrons in enzymatic and nonenzymatic reactions. ROS are, however, also destructive unless tightly controlled. Mammalian cells have developed a battery of defenses to prevent and repair the injuries caused by

Origin of ROS
Generation in mammalian organism Sources endogenous exogenous
prostaglandin synth. ultrasound respiratory chain smoke autooxidation radiation, cigarette drugs

HOONO
NO2. e-

.NO

OONO-

H+

O2 H2 O

.- e H O e .OH O2 2 2

e-

O2.-

O2 Fe2+

Fe3+

H+

FREE RADICAL S
phagocytes heat oxyhemoglobin pesticides oxidative enzymes infections

MyeloMyeloperoxidase

ClH2O

HOCl + Cl-

1O

Enzymatic sources of ROS

Xanthine oxidase Hypoxanthine + 2O2 pXanthine + O2.- + H2O2 pXanthine NADPH oxidase NADPH + O2 pNADP+ + O2.pNADP Amine oxidases R-CH2-NH2 + H2O + O2 pR-CHO + NH3 + H2O2 pR Myeloperoxidase Hypohalous acid formation H2O2 + X- + H+ pHOX + H2O pHOX NADH oxidase reaction Hb(Mb)-Fe3+ + ROOH pCompound I + ROH Hb(Mb)pCompound Compound I + NADPH pNADy+ Compound II pNAD Compound II + NADH pNADy+ E-Fe3+ pNAD EpNAD NADy+ O2 pNAD+ + O2.Aldehyde oxidase 2R2R-CHO + 2O2 p2R-COOH + O2.p2RDihydroorotate dehydrogenase Dihydroorotate + NADy+ O2 pNADH + O2.- + Orotic pNADH acid

Effect of superoxide dismutase (SOD) on reduction of cytochrome c by O2.- which is produced by xanthine oxidase (XO) in the presence of xanthine (X) no SOD
0.06 0.04
550 nm

Absorbance

XO 2 mU/ml
0.02 0.00

+ SOD 200 U/ml

-0.02 X100Qmol/l 0 100 200 300 400 time (s)
Institute of Experimental Pharmacology, SASc, Bratislava, SK

500

600

700

800

900

Nonenzymatic sources of ROS and autooxidation reactions

Fe2+ + O2 pFe3++ O2.pFe Hb(Mb)Hb(Mb)-Fe2+ + O2 p Hb(Mb)-Fe3++ O2.Hb(Mb)Catecholamines + O2 pMelanin + O2.pMelanin Reduced flavin Leukoflavin + O2 pFlavin semiquinone + pFlavin O2.Coenzyme Q-hydroquinone + O2 pCoenzyme Q pCoenzyme (ubiquinone) + O2 .Tetrahydropterin + 2 O2 pDihydropterin + 2 O2.pDihydropterin

Until the 1960s, free radicals were not 1960s, considered particularly relevant for mammalian physiology and pathology. pathology. The discoveries of the existence of superoxide dismutase (SOD) activity in mammalian cells in 1969 by McCord and Fridovich and association of bactericidal activity of neutrophils with production of the superoxide radical (O2.-) by Babior and coworkers in 1973, linked free radicals to 1973, numerous physiological and pathophysiological processes. processes. One decade later, in 1981, Granger and 1981, coworkers established a hypothesis on the role of these reactive species in the reperfusion injury after intestinal ischemia. ischemia.

resulting in a physiological balance between their production and elimination

ROS are tightly controlled

Enzymes: SOD (c, m) , (c, m) GPX (c, m), CAT (c, p) (c, m), (c, p) NonNon-enzyme antioxidants: vitamines (E,A,C), thiols, phenols, ceruloplasmin, transferrin, uric acid, albumin, etc.

ROS:
O2y, H2O2 , 1O yOH, HOCl 2

c-cytosolic, m-mitochondrial, pperoxisomal

Biological antioxidant defense mechanisms

Defense mechanisms in the organism
a. Catalytic free radical removal O2.- - spontaneous dismutation
- superoxide dismutase (SOD) - ceruloplasmin H2O2 - glutathion peroxidase (GTPx) - catalase (CAT) Organic hydroperoxides - GTPx Disulphide - GTPx Oxidised ascorbate - GTPx

O2.LPO

Fe2+

Fe3
+

b. Free radical scavengers (antioxidants) Vitamin E (E-tocopherol) O2.-, .OH,

LPO

.OH

H2 O2
GSH

Reduced ascorbic acid

in high concentrations of LPO Low m.w. thiols (e.g. cystein) Large m.w. thiols (e.g. albumin) (Fe)

O2.-, .OH,

c. Removal of Fe and Cu Ferritin, transferrin, lactoferrin

H 2O + O 2

GSSG

H2O

Under pathological condition

the physiological balance is lost

Enzymes: SOD,GPX,CAT NonNon-enzyme antioxidants: vitamines (E,A,C), thiols, uric acid, ceruloplasmin, transferrin, phenols, albumin, etc.

ROS:
O2y, H2O2 , 1O , 2 yOH, HOCl

Consequences are shown in the next panels

Disbalance between production and elimination of ROS develops during inflammation, ischemia/reperfusion, altered metabolism, action of drugs, pollutants, etc. Such disbalance causes pathology of brain, heart, vessels, gut, airways, muscle, parenchy- matous organs (liver, kidney, pancreas), eye, skin, joints, etc. Exposure of the tissues to ROS in a variety of biological systems has documented their ability to damage lipids, proteins and DNA. The resulting damage potentiated by increased free intracellular Ca2+ causes activation/deacti-vation of various

Mechanisms of ROS induced cell injury

LIPIDS Lipid SUGARS PROTEINS
Oxidation of thiols Carbonyl formation Damage to Ca2+ and other ion transport systems Amadori products Membrane damage Instability to maintain normal ion gradients Activation/Deactivation of various enzyme systems Depletion of ATP and NAD(P)(H) Poly ADP Altered gene ribosylation expression

DNA
DNA damage

Schiff bases peroxidation

AGEs
end products)

(Advanced glycation

Involvement of ROS in APOPTOSIS

NOXA (trauma, hypoxia
homeostatic under control to a limit

metabolic insufficiency
certain

activation of excitatory receptors) Ion disbalance caspase/calpaine ROS generation Mitochondrial failure BclBcl-2 / Bax disbalance

activation

CELL DEATH (necrosis / apoptosis)

Currently it is believed that free radicals are definitely paticipating in several health disorders. There are different pathologic conditions where extracellular, intracellular or both ROS act at least in part. However, in spite of the extensive studies our knowledge concerning the role and action of free radicals and ROS is still incomplete and changing.

Pathological conditions that may have a free radical component and sites of ROS actions
Hypo-, hyperoxygenation & Immune Reperfusion after Cataractogenesis reactions ischemia Chemical Radiation injury cancerogenesis FREE Ageing & Cancer senile dementia RADICALS Diabetes Atherosclerosis Iron, drug Parkinsonism & chemical Smoking, toxicity air Inflammator pollutants y & reactions drug induced reactions

intracellular

Intracellular & extracellular

extracellular

ROS generation during ischemia and reperfusion

ATP I S AMP Xanthine dehydrogenase C H Adenosine E Ca2+ proteases M Inosine I A Hypoxanthine+Xanthine oxidase O2.- activated chemoattractants REOXYGENATION Cl- H2O2 Circulating neutrophils MPO HOCl Fe2+
.OH

O2.-

Extravasated neutrophils

Fe3+ Neutrophil activators Chemoattractants

ROS in the sequence of events in STROKE

HYPOXIA
ATP depletion Cell depolarization (q Mg block of NMDA rec.) Excitatory aminoacid release Ca2+ influx into the cells Slow accumul. Ca2+ in mitochondria Activation of phosholipases, MPT pore opening in mitochondria proteinkinases, proteases, H+ gradient collapse in mitochondria endonucl., phosphatases etc. ROS generation ONOO- generation Devastatory effect in cells

NEURONAL DEATH
Therapeutic interventions: cyklosporine (specific MPT pore inhibitor), interventions: antioxidants (lazaroids, deferoxamine, SOD in liposomes, allopurinol) (lazaroids, allopurinol)

Frequent targets of ROS

gut

airways
HOCl

O2 y-

yOH

H2 O 2

brain & nerves

heart & vessel s

ROS affect different tissues and tissue components. They affect e.g. not only the smooth muscle cells, but also their epithelium, endothelium, innervation, membrane lipids, receptors, transmitter systems, prostanoid production, Ca2+ homeo- stasis, etc.)

Proposed mechanisms of ROS actions in airway

In physiological conditions In pathological conditions
O2.No changes

O2.-

Dominant contraction

H 2O 2 .OH

Dominant contraction

H2O2

Long-lasting contraction

.OH
Dominant relaxation

Intensive contraction

Late Resting Initial change change tone of tone of tone after ROS after ROS

epithelium smooth muscle

Late Resting Initial change change tone of tone of tone after ROS after ROS

SOD- superoxide dismutase; Cat catalase; LMWAO low molecular weight antioxidants

Institute of Experimental Pharmacology, SASc, Bratislava, SK

reacting with O2y- gives rise to unstable peroxynitrite, which decom-poses also to the most toxic yOH. Because of the large energy gain of the reduction of yOH to H2O, this radical reacts instantaneously with any biological molecule in its immediate environment by abstracting hydrogen atom.
yNO

Production of ROS in endothelium and neutrophils

yOH

OONOO
2

l-arginine e n d o t h e l i u m
NOS

O2 ATP

NADPH

yNO

yNO

AMP adenosine hypoxanthine O2 XO uric acid

yOH

O2 y H 2O 2 MPO HOCl

NADPH NADPH oxidase

yOH

NADP+

inosine XDH

O2
H 2O 2

y-

MPO

n e u t r o p h i l

Institute of Experimental Pharmacology, SASc, Bratislava, SK

Diseases that may have ROS related pathogenesis Airways I

Normobaric hyperoxic injury Bronchopulmonary dysplasia Idiopathic pulmonary fibrosis Respiratory distress syndromes (ARDS, IRDS) Emphysema Chronic bronchitis & asthma bronchiale Asbestosis Inhaled pollutants, smoke, chemicals (e.g. paraquat, bleomycin) & oxidants (e.g. SO2, NOx, O 3) Ischemia/reperfusion Crohns disease Ulcerative colitis & necrotizing enterocolitis Gastric & intestinal ulcers Chemicals (e.g. NSAID)

Gut

Heart and vessels

II
infarction,

Ischemia/reperfusion (after transplantation) Chemicals (e.g. ethanol, doxorubicin) Atherosclerosis/hypertension Selenium deficiency Vasculitis

Brain and nerves

Hyperbaric hyperoxic injury Parkinson's disease Alzheimers disease (details see in the next panel) Amyotrophic lateral scleroses Neuropathies (e.g. diabetic) Neurotoxins (e.g. 6-hydroxydopamine, MPTP) Vitamin E deficiency Neuronal ceroid lipofuscinoses Traumatic injury/hemorrhage/inflammation Ischemia/reperfusion HIV-dementia Multiple sclerosis

ALZHEIMER DISEASE and oxidative stress

o Protein oxidation (carbonyls) - crosslinking o Fe in neurons with fibrilary aggregates (Xhyperphosphoryl.protein) o Content of aluminium in neurons with fibrilllary aggregates F-amyloid generation (direct cytotoxic action,o Cai, generation of action,o ROS even in the absence of Me2+) o Activity of microglia (brain macrophages = ROS source) q Activity of CAT without q SOD activity resulting in o H2O2 and oyOH Generation of lipid hydroperoxides and reactive cytotoxic aldehydes (e.g. HNE) Therapeutic interventions: antioxidants and ROS scavengers interventions:

Blood

III

Chemicals (e.g. phenylhydrazine, primaquine, sulphonamides, lead) Protoporphyrine photooxidation Malaria Anemias (sickle cell, favism)

Liver
Ischemia/reperfusion Chemicals (e.g. halogenated hydrocarbons, quinones, ethanol, acetaminophen) Accumulation of iron or copper Endotoxin

Kidney

Autoimmune nephrosis (inflammation, e.g. glomerulonephritis) Chemicals (e.g. aminoglycosides, heavy metals)

Pancreas Eye

IV

Acute & chronic pancreatitis Diabetes mellitus Retinopathy of prematurity Photic retinopathy Cataracts Laser photoablation

Skin
Radiation (solar, ionising) Thermal injury Chemicals (photosensitizers, e.g. tetracyclines) Contact dermatitis Porphyria

V
Muscle
Muscular dystrophy Multiple sclerosis Exercise

Others
Aging Pregnancy and newborn complications Radiation injury Cancer Chemicals (e.g. alloxan, iron overload, radiosensitizers) Autoimmune diseases (e.g. rheumatoid arthritis, lupus erythematodes) Inflammation (in general)

Potential antioxidant therapy I

Inhibitors of ROS synthesis
NADPH-oxidase Inhibitors Flavoprotein inhibitors (FAD analogs, antibodies of cytP450 reductase) Agents forming complexes with Fe2+ in cyt b (butylisocyanide, imidazole, pyridine) Mg2+(enabling FAD binding),Fe2+ ,Cu2+ chelators (bathophenantroline, EDTA, EGTA, deferoxamine, bilirubin) Thiol reagents (N-ethylmaleimide, 1-naphtol, 1,4naphtoquinone) NADPH analogs (NADPH 2,3-dialdehyde) Inhibitors of metabolism of AA and PLA2 IMAO (Deprenyl) Others (corticosteroids, diphenyliodonium)  Inhibitors of xanthine oxidase (tungsten, oxypurinol,


Agents supporting and complementing enzymatic protective systems

dismutase (SOD) SOD (Lip-SOD,PEG-SOD) Copper diisopropylsalicylate SOD mimetics  Catalase (Cat) Cat (Lip-CatTP, Peg-CatTP)  Glutathionperoxidase (GTPx) GSH, GSH methylester, GSH diethylmaleate Low m.w. thiols (e.g. cystein) High m.w. thiols (e.g. albumin) L-2-oxothiazidolidine-4-carboxylate N-acetylcysteine Ebselen Selenium  Lactoperoxidase & DT-diaphorase
 Superoxide

II

Drugs interfering with iron and copper metabolism

(deferoxamine, hemopexine, ferritin, transferrin, lactoferrin, ceruloplasmin, serum albumin)

III

Antioxidants
 

    

Vitamins and their analogues (vitamin E, vitamin C, carotenoids, oxycarotenoids) Phenol derivatives (eugenol, guajacol, probucol, N,Ndiphenylphenylendiamine) Flavone derivatives (flavonoids, isoflavonoids, allirazine, green tea) Indol derivatives (stobadine, carvedilol, melatonin, Kcarbolines) Xanthine derivatives (allopurinol, oxypurinol, uric acid) 21-amino steroids (lazaroids) Antiinflammatory drugs (piroxicam, flufenamic acid, mefenamic acid, hydroquinone, sulindac, fenylbutazone,

IV
 Agents

containing sulfur (cysteine, cysteamine,

GSH, dithiothreitol, N-acetylcysteine, ACE inhibitors, dimethylthiourea, thiourea, thiomalate, hypotaurine, taurine, penicillamine, 2-amino-2-thiazole, dihydrolipoate, E-mercaptopropionyl glycine, N-2mercaptopropionyl glycine, F-mercaptoethanole, D,L-methionine, other low and high m.w. thiols) .  Nitroso compounds ( NO, nitrosopine)  Other drugs (F-adrenolytics, H2-antihistaminics, calcium channel blockers, pentoxyphylline, carbanilates, urea, bilirubin, glucans, manitol, glucose, 2-methylaminochromans, DMSO,

V
Inhibition of O2.- formation
 Nonsteroid antiflogistics

Antiasthmatics (Fadrenomimetics, corticoids, methylxanthines)  Prostaglandins  Flavonoids  Antibiotics (e.g. minocycline)  Antimalarics  Inhibitors of ACE  Dipyridamol


VI/a
Scavenging or removal of ROS  Scavenging of generated O2.Flavonoids & other natural products Vitamins E, C, A(Fcarotene) Synthetic analogs of PGB2 Dipyridamol Pentoxiphylline Antibiotics .NO donors 5-acetylsalicylic acid Uric acid

 Scavenging  Scavenging
1O 2

HOCl or quenching of

Uric acid Taurine, hypotaurine Silymarine F-carotene Vitamin E Stobadine

VI/b
Scavenging or removal of ROS
 Removal of H2O2 Catalase (not working in the presence of .NO) N-acetylcysteine  Elimination of OH. Manitol Thiourea Stobadine Melatonin Probucol 5-acetylsalicylic acid Lazaroids DMSO, DMTU, BHT Uric acid Glucose

Scavenging or removal of ROS  Lipid oxidation chain breaking antioxidants (anti LO. and LOO.)
Bilirubin Vitamins E Vitamin C F-carotenoids and oxycarotenoids Stobadine Melatonin E-lipoic acid Uric acid Lazaroids BHT, BHA Ehoxyquin Some positive2-methylaminochroman clinical studies with results from preclinical and the pyridoindole STOBADINE, which possesses significant antioxidant, mainly hydroxyl radical scavenging, lipid oxidation chain breaking and singlet oxygen quenching properties, as an

VI/c

Therapeutic relevance of the use of antioxidants DISEASE ANTIOXIDANT THERAPEUTIC SUCCESS I

Cardiovascular Carotenoids Ascorbic acid Tocopherols Selen Probucol Flavonoids Penicillamine Tocopherols SOD, SOD+CAT Lipoic acid Allopurinol Tocopherols Deferoxamine SOD, SOD+CAT Tocopherols Ascorbic acid Carotenoids s + s + s + + s s s + + + + + +

Newborn hypoxia induced injuries Ischemia/Reperfusion (of the heart, brain, gut, kidney) Transplantation and tissue preservation

DISEASE

ANTIOXIDANT

II

THERAPEUTIC SUCCESS

Intravascular hemorrhage Platelets aggregation

Tocopherols Ascorbic acid Flavonoids Stobadine Hemochromatosis Deferoxamine Head trauma Lazaroids (details see in the next Stobadine panel) derivatives Phenyl-butylnitones Subarrachnoidal Lazaroids hemorrhage Respiratory distress SOD syndromes (IRDS, Allopurinol ARDS) Tocopherols Bronchial asthma SOD+CAT Thiols Tocopherols Pulmonary injuries N-acetylcysteine Thiols

+ + + + ++ s + + s + + + sss+ +

ROS in the sequence of events in NEUROTRAUMA

TRAUMA Excitatory aminoacid release (GLU) Ca2+ influx into the cells cascade Protease/lipase activation Cell depolarization activ.) (q Mg block of NMDA rec.) Na+influx Edema DEATH TRIAD : EXCITOTOXICITY, Ca-OVERLOAD, OXIDATIVE STRESS EXCITOTOXICITY, Ca-OVERLOAD, Therapeutic interventions: -SH donors (N-acetylcysteine), lazaroids, interventions: (Nsteroids, deferoxamine, SOD, vitamines A,E,C, Activ. of inflam. (PAF, eikosanoids, cytokines, PMN ROS generation cell devastation NEURONAL

DISEASE

III

ANTIOXIDANT

THERAPEUTIC SUCCESS

Flu (cold) Retrolental fibroplasia Cataract Inflammatory diseases of the gut (IBD) Hepatopathies

Ascorbic acid Tocopherols Tocopherols 5-aminosalicylates Sulfasalazine Sulfapyridine SOD+CAT Glucans Lipoic acid Silymarin Stobadine N-acetylcysteine Glutathione Deferoxamine Carotenoids Tretionin Carotenoids

s + + + + + s s s s + ++ + + ++ + ++

Paracetamol intoxication Chemical poisonings Photosensibilization UV irradiation

DISEASE

IV

ANTIOXIDANT

THERAPEUTIC SUCCESS

Rheumatoid arthritis Parkinsomism Wilsons disease Cerebro-vascular spasms Cancer

SOD Penicillamine Deferoxamine Tocopherols Penicillamine Carotenoids Tocopherols Thiols SOD+CAT Carotenoids Ascorbic acid Tocopherols Selen Flavonoids Thiols

s + s s ++ s s s s ss s s s

Conclusions I

ROS act by:

increase of membrane lipid peroxidation increase of prostaglandin production increase of intracellular free calcium alteration of conductivity of ion channels alteration of enzyme activity alteration of release/action of neurotransmitters reduction of half-life of biologically active substances damage of proteins damage of DNA, genes and protein Mechanisms of ROS action differ in various biological tissues. synthesis Their actions dependcarbohydrates of the tissue itself, the damage of upon condition corresponding epithelium, endothelium, innervation, etc.

Conclusions II The effects of ROS could be prevented or stopped by:

- elimination of undesirable physical and chemical influences - protection of tissues from chronic inflammation - protection of tissues from ischemia

reduction of their generation

their elimination

- substitution with antioxidant enzymes - substitution with non-enzyme antioxidants and scavengers - protection of cells from intracellular free calcium accumulation and its effects

interaction with their effects

Conclusions III Therapeutic success with the use of antioxidants, quenchers and scavengers
There are Promising clinical results
e.g. in photosensibilization, paracetamol intoxication, hemochromatosis

Controversial clinical results

e.g. in ischemia/reperfusion, subarachnoidal hemorrhage, respiratory distress syndromes

Minimal therapeutic effects

e.g. in asthma bronchiale, cancer

tRNA is transfer RNA that carries an a.a to the mRNA to be incorporated into the peptide chain.  mRNA is a type of RNA that encoding the sequence of the protein in the form of a trinucleotide code . The specific sequence of the nucleotide is accomplished through transcription.


PROTEIN CATABOLISM
Has various indication:  Comprises of Digestion and Absorption  Is carried out via proteolysis  is the directed degradation (digestion) of proteins (digestion) by cellular enzymes called proteases (various kinds) releasing peptide/A.A  The digestion of proteins from foods as a source of amino acids (aas)  The aas constituting aa pool are metabolized further ( aa catabolism)

Deamination is also an oxidative reaction occurs under aerobic conditions in all tissues but especially the liver. During oxidative deamination, an amino acid is converted into the corresponding keto acid by the removal of the amine functional group as ammonia and the amine functional group is replaced by the ketone group. The reaction is catalysed by glutamate dehydrogenase which is allosterically controlled by ATP and ADP. ATP acts as an inhibitor whereas ADP is an activator.

The ammonia eventually goes into the urea cycle. Oxidative deamination occurs primarily on glutamic acid because glutamic acid was the end product of many transamination reactions.

GD

The glutamate dehydrogenase (GD) is allosterically controlled by ATP and ADP. ATP acts as an inhibitor whereas ADP is an activator.

Summary of Urea Cycle  Occurs in liver cells  Is a 5 steps cycle: 1 step in mitochondria 4 steps in cytosol  Main substrates: NH3, CO2 and Aspartate.  In the matrix of mitochondria occurs CPS I and OTC catalysed rxn, CPS rxn uses 2ATP and reaction is irreversible  Citrulline Ornithine occur in cytosol, in 4 steps -Citrulline is tranported across the inner membrane by a carrier neutral aa. - enzymes are arginosuccinate synthase, arginosuccinate lyase and arginase  Urea transferred to kidney through blood and excreted as urine

CANCER DRUGS AND THEIR DEVELOPMENT: A PRIMER FOR LIFE SCIENTISTS

Edward A. Sausville, M.D., Ph.D. Marlene and Stewart Greenebaum Cancer Center School of Medicine University of Maryland

TYPES OF CANCER TREATMENTS  Surgery

Radiation  Chemotherapy: "Cytotoxic"  Chemotherapy: "Targeted"

  

Growth Factor Dependent pathways Modulate apoptosis AntiAnti- angiogenic

Biological Therapies : Enlist at least partially host mechanisms  Immunotherapy (antibodies)  Targeted Toxins  Tumor Vaccines  Tumor directed gene therapy  Tumor Directed Cell Therapy Immunologic Non Immunologic

CANCER DRUGS:
HOW DO WE KNOW WE Treatment A HAVE A - PHASE III CLINICAL TRIALWINNER?
= WINNER %
Alive Treatment B or no R x Time

- PHASE II = POTENTIAL WINNER

; Time?

R x
- PRECLINICAL MODEL

(e.g., mouse or rat)

Tumor Size

Rx

Untreated Cytostatic Cytotoxic Time

CANCER CHEMOTHERAPY: ORIGINS OF CONCEPT-I CONCEPT

Paul Ehrlich, 19th cent German Pathologist:



Different tissues / parts of cells take up different chemical dyes differentially, e.g. hematoxalin and eosin staining Lead to hypothesis that chemicals could be defined with selective affinity for disease components: micro-organisms, microtumor cells, etc. Popularly characterized as "Magic Bullets"

During WWI, accidental release of mustard gas in industrial accidents and in afflicted soldiers: bone marrow toxicity


Lead Alfred Gilman and other to propose use of mustad gas derivatives to treat hematological neoplasms

CANCER CHEMOTHERAPY: ORIGINS OF CONCEPT-II CONCEPT

Biosynthetic understanding of nucleic acids and proteins suggest precursors might be amenable to derivatization to prevent processes important in cell growth
 

Farber: antifolates Hitchings and Elion: purines

Natural extracts (plants, bacteria) define substances with antiproliferative activity for bacteria and / or mammalian cells
 

AntiAnti-tumor antibiotics AntiAnti-mitotic agents (colchicine-like molecules) (colchicine-

CURABILITY OF CANCERS WITH CHEMOTHERAPY

Sausville & Longo in Harrisons Principles of Internal Medicine, 2001

CHEMOTHERAPY CANCER CELL KILLING MECHANISMS



Historical: Cytokinetic features as key basis for success:  Phase specific / non-specific agents nonModern: Consideration of Cancer Chemotherapeutic Agents as Activating Cell death pathways

KINETIC PARAMETERS OF SOME HUMAN TUMORS

LOCATION OF DRUG ACTION IN THE CELL CYCLE

CELL CYCLE SPECIFICITY OF EFFECT

Cell Cycle Non-Specific Agents: Exponential Surviving Fraction vs Dose Cell Cycle Phase-Specific Agent: Plateau Surviving Fraction vs Dose Rationale = Insensitive Fraction
Surviving Fraction Tumor Cells

Single Dose in Terms of LD10

Distinguish between cell cycle specific killing vs cell cycle point of arrest A late G2 block characterizes drugs which alter DNA integrity either by cross-linking or breakage

EFFECTS OF DOXORUBICIN ON THE DNA HISTOGRAM IN SARCOMA 180 IN VITRO

A Pretreatment Frequency

B 8 hrs

C 16 hrs

D 24 hrs

Cell DNA Content

Cells progress from the G1 and early S region (A and B) and accumulate in the G2M region of the histogram (C and D).

LOGLOG-KILL HYPOTHESIS
 Constant  1012

fraction of cells are killed.

108

CUMULATIVE MORTALITY IN ANIMALS RECEIVING ONE TO FOUR COURSES OF ARA-C (IP L1210 LEUKEMIA)

Cumulative % Mortality

8 X 105 CELLS

8 X 106 CELLS

KEY CONCEPT: IN IMMUNO-INTACT ANIMAL CHEMO WORKS BETTER WITH SMALLER TUMOR BURDEN

DRUG RESISTANCE
degradation of drug  Altered affinity of target enzyme  Changes in metabolic activation  Altered drug transport  Gene amplification  DNA repair  Increase thiol content  Glutathione increase (H2O2; free radicals)
 Excessive

Cytokine/hormone death signal TNF-RI INTEGRATION FASR TRAILR OF CELL

Chemotherapy agent Membrane damage Ceramide Reactive radicals AIF ? Bax AIF DNA Damage

DEATH RESPONSES
FADD

Sphingomyelinase FLICE AKT P-Bad Bcl2 Bad Effector caspases Caspase 3

Caspase 8

(Mitochondrial Damage)

+ +
Cytochrome APAF Caspase 9 p53 Proapoptotic gene expression

Specific cell protein targets

Nuclease activation DNA digestion

Sausville & Longo in Harrisons Principles of Internal Medicine, 2001

GOALS OF PRECLINICAL DRUG STUDIES Regulatory framework

IND = Investigational New Drug application = approval by FDA to conduct human studies; main criterion : SAFETY AND LIKELY REVERSIBLE TOXICITY = allows start of Phase I trials NDA = New Drug Application = basis for sale to public; main criteria: SAFETY AND SOME MEASURE OF EFFICACY = result of Phase II/III trials

EMPIRICAL DRUG SCREENDISCOVERY PHARMACOLOGY

BIOLOGICAL ACTIVITY (in vitro/in vivo)

CHEMISTRY

OPTIMIZED SCHEDULE (in vivo) IND-DIRECTED TOX/FORMULATION PHASE I: DOSE/SCHEDULE HUMAN PHARM/TOX PHASE II: ACTIVITY PHASE III: COMPARE WITH STANDARD

COMPONENTS OF AN IND
The goal of the pre-clinical process Form 1571 Table of Contents Intro Statement / Plan Investigator Brochure Clinical Protocol Chemistry, Manufacture, Control Prior Human Experience Additional Info - Data monitoring, Quality Assurance Pharmacology/ Toxicology

CLINICAL DEVELOPMENT OF ANTICANCER DRUGS [DeGeorge, et al, Cancer Chemotherapy Pharmacology The types of preclinical studies expected for support of clinical trials and (1998)new drug depends on both 41: 173-185] 173marketing of a
the intended use of the drug and the population of patients being studies and treated. In situations where potential benefits are greatest (Advanced, lifethreatening disease), greater risks of treatment toxicity can be accepted and the required preclinical testing can be minimal.

PRECLINICAL PHARMACOLOGY STUDIES FOR ANTIANTI Development of Sensitive Analytical Methods for NEOPLASTIC DRUGS Drugs in Biological Fluids & Tissues
     

Determine In Vitro Stability and Protein Binding Determine Pharmacokinetics in Rodents (& Dogs) Identification and Analysis of Metabolites Define Optimal Dose Schedule and Blood Sampling Times Define CP and/or AUC with Efficacy, Safety & Toxicity Analog Evaluation - Determine Optimal Development Candidate

ARE PRE-CLINICAL PREPHARMACOLOGY STUDIES REQUIRED?



NO! There are numerous examples where the compound cannot be assayed at efficacious doses: -rapid learance -metabolism -very potent compound / insensitive assay

Natural product extracts could conceivably have activity without definable single molecule basis for activity BUT  In modern times, these should be exceptional cases


OBJECTIVES OF PRECLINICAL TOXICOLOGY STUDIES FOR ANTIANTINEOPLASTIC DRUGS  DETERMINE IN APPROPRIATE ANIMAL MODELS:


The Maximum Tolerated Dose (MTD) Dose Limiting Toxicities ( DLT ) ScheduleSchedule-Dependent Toxicity Reversibility of Adverse Effects A Safe Clinical Starting Dose

FDA PRECLINICAL PHARMACOLOGY & TOXICOLOGY DRUGS REQUIREMENTS

 

Two Species - Rodent & Non-rodent NonClinical Route & Schedule

Follow NCI Guidelines

Pharmacokinetics - Optional

BIOLOGICALS
 

Most Relevant Species Clinical Route & Schedule

BENZOYLPHENYLUR EA PRECLINICAL MTD & DLTs

Schedule q4Dx3, po MTD
(Total Dose)

RAT 360 mg/m2

Bone Marrow GI Tract

DOG > 150 < 240 mg/m2

Bone Marrow, GI Tract

DLT

Starting Dose: 24 mg/m2

RATIONAL DRUG DISCOVERYPHARMACOLOGY MOLECULAR TARGET SCREEN

Biochemical Engineered cell Animal (yeast/worm/fish) (to affect target)

CHEMISTRY

TARGET-DEPENDENT IN VIVO MODEL IND DIRECTED TOX/FORM PHASE I: DOSE/SCHEDULE: HUMAN PHARM/TOX; ? AFFECT TARGET PHASE II: ACTIVITY = ? AFFECT TARGET PHASE III: COMPARE WITH STANDARD; STRATIFY BY TARGET?

Targeted Cancer Treatments

Cytotoxic Cancer Treatments

Reversible Biochemistry
Altered target function

Covalent Pseudoirreversible OR Biochemistry Pharmacology

Dormancy / Diferentiation OR Cell Death Program OR Microenvironment

Lesion triggers cell death as consequence of Drug induced lesion, assuming no repair

WHAT IS A MOLECULARLY TARGETED AGENT vs. A CYTOTOXIC? Cytotoxic Targeted Agent

land on target organism toxicity therapeutic effect

% Effect

% Effect

land on target Target-related Toxicity?

organism therapeutic effect toxicity

Dose or Concentration Elicit useful effect by cells response to drug landing on target

Dose or Concentration Elicit useful effect by altering target function or position in cell

ZDZD-1839 (Gefitinib; Iressa): AN EGF-R PROTEIN KINASE EGFANTAGONIST

F O N O N Me O N NH Cl

Phase I: 14 d on / 14 d off; 50 - 700 mg (MTD) Long t (46 hr) in humans Toxicities: diarrhea (50 - 60%); cutaneous (30 - 40%) 4 / 64 PR (NSCLC); 2 with > 2 mo

OSI-774 (Erlotinib: Tarceva): OSIAN EGF-R PROTEIN KINASE EGFANTAGONIST with ATP Inhibition of purified EGFR kinase Competition
[OSI-774] % of Control ( S.D.) Phosphorylation 1/ V OSI-774 (nM) 1 / [ATP] (1/ QM)
NH O O O O N N HCl

Moyer et al, Cancer Res 57: 4838, 1997

OSIOSI-774: AN EGF-R PROTEIN KINASE EGFANTAGONIST

Pollack et al, J Pharmacol Exp Ther 291: 739, 1999

GEFITINIB THERAPY >200 mg/d INHIBIT ACTIVATED MAPK

Pre-Gefitinib
Interfollicular epidermis

On-Gefitinib
% Activated MAPK positive 25 20 15 10 5 0 Pre

p < .001

Hair follicle

On

Albanell et al, J Clin Oncol 20: 110, 2002

IDEALIDEAL- 1 AND IDEAL 2

Efficacy and safety of Gefitinib in NSCLC post relapse from Platinum based chemotherapy IDEAL 1 (N = 209) IDEAL 2 (N= 216)
250 mg/m2 -% Tumor response 18.4 -% Tumor control -% Symptom improvement -Duration (mo) -Overall survival (mo) 54.3 40.3 500 mg/M2 19 51.4 37.7 250 mg/m2 11.8 42.2 43.1 500 mg/M2 8.8 36.0 35.1

3.2 7.6

5.1 7.9

NA 6.1

5.4 6.0

PATIENTS RESPONDING TO IRESSA HAVE FREQUENT EGFEGF-R MUTATIONS

NEJM, 2004

IRESSA-RESPONDING MUTATIONS IRESSAARE ACTIVATING MUTATIONS

NEJM, 2004

OTHER DETERMINANTS OF TKI RESPONSE

AKT-status: Bianco et al Oncogene 22, 2812, 2003 A431 MDA468 PTEN + EGFR + + IC50 Gef <0.1 uM >2 uM EGFR-YP sens sens MDA/PTEN : MDA/vector <0.1/~1 uM Insulin-like Growth Factor (IGF) Receptor: Lu et al J. Natl Cancer Inst 93 1852 * resistance to Herceptin? Ras allele status: 20% of lung CA population mutated; preliminary results suggest ~0% chance of response to Tarceva

DIFFERENT CLINICAL TRIALS :DIFFERENT GOALS -I Phase I: Clinical Pharmacology /

pharmacokinetics / pharmacodynamics / toxicity 1st use of a new agent or combo in humans Advanced disease (not single tumor type) No established Rx / failed established Rx Phase II: Initial investigation of activity. Assess efficacy, feasibility, toxicity Specific tumor type Cytotoxic Rx: activity assessed by response (shrinkage of measurable disease) Biologics / Targeted Rx: either metastatic or adjuvant: activity assessed by response or time to progression.

PHASE I PROCESS-1 PROCESS Phase I Process:

Define schedule to be studied (animals) Define starting dose (animals) Administer to patients with a variety of tumor types UNLESS compelling rationale for a selected population (e.g., antibody targeting specific cell surface receptor) Classic dose escalation schedule Treat three patients / dose level Wait until all patients complete dose level Grade toxicity: (NCI CTC, WHO, etc) Grade 0 : No toxicity Grade 1: Adverse event not require Rx Grade 2: Adverse event Rxable Grade 3: Potentially Life threatening or cause hospitalization Grade 4: Actually Life threatening Grade 5 Cause death http://ctep.nci.nih.gov

PHASE I PROCESS-2 PROCESS Phase I Process:

Can escalate dose if 3 pts complete dosing with <Grade 3 non hematopoetic; <Grade 4 hematopoetic If 1/3 have Gr 3, increase cohort to 6 If 2 of 6 pts have Gr 3, maximum tolerated dose exceeded and go to lower dose level; MTD and Recommended P2 dose is where 1 of 6 have Gr 3 toxicity Obtain Pharmacology on all levels (suggest dose / response basis for toxicity) Obtain evidence of drug effect on intended target at MTD (pharmacodynamics) Follow for evidence of response: If pts have stable disease or better continue treat until progression of disease If evidence of progression of disease with q 2 3 month imaging, come off Rx If tolerate poorly, come off Rx

UNEXPECTEDADVERSE EVENTS
Any adverse drug experience (AE), the specificity or severity of which is not consistent with the current investigator brochure; or, if an investigator is not required or available, the specificity or severity of which is not consistent with the risk information described in the general investigational plan or elsewhere in the currently application, as amended. 21 CFR 312 32

SERIOUSADVERSE EVENTS
Any adverse drug experience occurring at any dose that results in any of the following outcomes: Death Life-threatening: -Inpatient hospitalization or prolongation of existing hospitalization -Persistent or significant disability/incapacity -Congenital anomaly/birth defect 21 CFR 312.32

TOXICITY
Not clearly defined in the Code of Federal Regulations (CFR) .International Conference on Hatmonization (ICH) term is adverse drug reaction An adverse event that has an attribution of possible, probable or definitely related to investigational treatment. Causal relationship between the investigational product and the adverse.

INVESTIGATOR RESPONSIBILITIES and An investigator is required to prepare

maintain adequate and accurate case histories that record all observations and other data pertinent to the investigation on each individual administered the investigational drug or employed as a control in the investigation. (21 CFR 312.62) Adverse events which are severe, unexpected, and related to the new drug need to be reported to supervising bodies (IRB, FDA) expeditiously

MSMS-275 (NSC#706995) A NEW HISTONE DEACETYLASE INHIBITOR

In vitro cell lines

 

In vivo xenografts


NCI cell line screen unique Promotes gene expression favoring growth arrest and differentiation

Mitsui: gastric, epidermoid, pancreatic, colon, ovarian, and NSCL NCI: myeloma and SCLC

Mitsui Pharmaceuticals. Saito et al., PNAS 96:4592, 1999.

TRANSCRIPTIONAL CONTROL
Co-activators

General transcription factors

HAT

Activator
Ac Ac Ac

Nucleosome Repressor
HDAC

Co-repressors

Muratani and Tansey, Nat Rev Mol Cell Biol 4: 192, 2003

MSMS-275 Initial Findings: Daily x 28 dosing (N = 2) DLT at 2  2/2 patients experienced

mg/m2/d


DLTs
Abdominal/epigastric pain, hypophophastemia, infection, pleural effusion, atrial fibrillation, LFTs and hyponatremia. All toxicities appear reversible

MTD exceeded

MSMS-275 t is possibly longer in humans than animals  Increased histone acetylation observed after one dose of MS-275 (1/10th of rat MSMTD)


Q 14 Day Dose Escalation

Level
1 2 3 4 5 6

Dose (mg/m2)
2 4 6 8 10 12

Number of Patients
3 3 6 5 6 5

Traditional cohort and no intra-patient dose escalation, intraadministered with meal.

MS 275: Dose Limiting Toxicities at Each Level Dose level # of patients Toxicities

1 2 3 4 5 6

0/3 0/3 1/6 0/5 1/6 2/5

0 0 3* 0 3 ** 7 ***

* Anorexia, nausea, vomiting ** Anorexia, nausea and vomiting *** Anorexia, nausea, vomiting, and fatigue

MSMS-275: > 3 cycles Adverse Events (Q14 Day)

 Cumulative
 

dosing intensify AEs > cycle 3

dose reductions decrease dosing frequency

 The
   

frequent cumulative AEs are:

Hypoalbuminemia Fatigue Neutropenia Thrombocytopenia

MS-275 Response/Length on Study

6
Days

on study: 11-300 days 11PR or CR

5 4 3 2 1
Dose 0 Level

No

15

SDs (62-300 days) (62-

100

200

300

Days

MSMS-275: Representative ConcentrationConcentration-Time Profile

dose=10, Patient=18 Rsq=0.8547 Rsq_adjusted=0.8256 HL_Lambda_z=60.2458 (7 points used in calculation) Uniform Weighting

100

10

Observed Predicted

1 0 10 20 30 40 50 60 70 80 90 Time (h)

MSMS-275: Dose-Proportionality DoseEvaluation: Cmax

40

(ng/mL)
max

30

20

MS-275 C

10

0 0 2 4 6
2

10

12

Dose (mg/m ) (Mean Values s SD)

MSMS-275: Summary of Pharmacokinetic Data Parameter Mean 22) Range (n =(SD)

CL/F (L/h/m2) T1/2 (h) Tmax (h) MRT (h) 16.9 (5.66)* 49.0 (15.0) 2.0** 71.0 (21.0)*** 7.15 27.3 23.3 73.0 0.50 60 31.3 103

* P = 0.048 (Kruskal-Wallis & Tukey-Kramer: 4 mg/m2 versus 8 mg/m2) ** Median value *** P = 0.55 (Kruskal-Wallis one-way ANOVA on ranks)

Peripheral Blood Mononuclear Cell Histone H3 Hyperacetylation in Response to MS-275 MS-

Pre

6h

48 h

96 h

Patient A (8 mg/m2)

Pre

6h

48 h

96 h

Patient B (8 mg/m2)

MS-275 Induced PBMC Histone H3 Hyperacetylation

6

0 0h 6h 12h 24h 48h 96h 14d

Time

H s t n e A ce t a ton ( o d -n c rease ) i o yl i F l I
02 04 06 08 02 5

PR E 8h 12 h 24 h we ek 2 3 4 5 6 7 ek ek ek ek ek we we we we we

emit tnemtaerT

H s t n e A ce t a ton ( o d -n c rease ) i o yl i F l I
01 51

PR E 8h 12 h 24 h we we we we we we ek ek ek ek ek ek 2 3 4 5 6 7

emit tnemtaerT

3H enotsiH

4H enotsiH

Progressive Acetylation of Histones H3 and H4 in Patients PBLs After Treatment with MS-275

MS 275- Phase I Conclusions 275 Q14

Day schedule is tolerated better than

QD
 

DLT: Nausea, vomiting anorexia and fatigue MTD: 10 mg/m2

 PK


suggests the possibility of more frequent dosing than q14 day

A once weekly x 4 every 6 weeks schedule is on going.

 Consider

IV formulation
Ryan et al. J. Clin. Oncol. 23: 3912, 2005

BENEFIT FROM PHASE 1 ONCOLOGY TRIALS?

Traditional view: Patients never benefit; just toxicity defining Review of NCI sponsored P1 trials 1991-2002: In 10,402 patients, CR and PR 10.6% Higher (17.8%) when combine new agent with standard agent; single new agent 4.4% Stable disease > 2 mo in 34% Overall mortality due to toxicity: 0.49%
Horstmann et al. NEJM 352: 895, 2005

OVERVIEW OF PHASE II DESIGNS

Single stage: Treat preset group to define level of activity for further testing Two Stage: Treat a small number of patients and if not observe activity above a certain level, close. Randomized Phase II: Not intended to be comparative for efficacy of treatment in disease; powered to detect likely similarity between treatments. Suggest basis for proceeding to formal Phase III Rare disease / response to standard therapy insufficiently documented / heterogeneity of prognostic factors / test multiple new therapies simultaneously

DIFFERENT TRIALS :DIFFERENT GOALS - II

Phase III: Full Scale comparative treatment between new teatemtn and standard of care. Randomized : Progress or emergence of differences monitored by a Data Safety Monitoring Committee Stratified Standard of care may be no treatment (historically difficult in oncology, as is placebo containg trial) Phase IV: Post marketing Long term effects of treatment Satisfy requirement for conversion from accelerated approval to full approval

Example of P3: Tamoxifen 5 Years vs No Recurrences Treatment Breast Deaths

100
85.2 76.1

100
89.5 76.8 64.9 69.4% 7.4 (SE 1.1) 57.0% 7.9 (SE 1.5)

80
73.7 11.5 (SE 0.9) 62.7 13.4 (SE 1.1)

68.2

80

86.3% 3.2 (SE 0.7)

68% Percent
54.9 13.4 (SE 1.4)

Percent

60

55%

60

65% 57%

40

20

ER+ Actuarial estimate and SE Allocated tamoxifen Allocated control

40

20

ER+ Actuarial estimate and SE Allocated tamoxifen Allocated control

0 0 5 10 15

0 0 5 10 15

Years
Early Breast Cancer Trialists Group. 2000.

Years

REQUIREMENTS FOR DRUG APPROVAL

21 CFR 314.126: Reports of adequate and well controlled 1 investigations provide the primary basisfor determining whether there is substantial evidence 2 to support claims of effectiveness for new drugs and antibiotics. 1: Could be placebo controlled, no-active treatment controlled, comparison of doses, historical comparison. 2: Two adequate and well controlled trials although in some cases one trial with supporting studies may be sufficient.

RELEVANT ONCOLOGY CLINICAL TRIALS ENDPOINTS

Choice of end-point depends on:

Type of drug (cytotoxic / hormonal / biologic) Type of tumor and patient population Phase of the trial Type of approval sought: Traditional unlimited vs. accelerated

TRADITIONAL APPROVAL = AVAILABLE IN THE CORNER PHARMACY WITHOUT Clinical benefit endpoints:REVIEW SUBSEQUENT

Survival (Overall Survival, Progression Free Survival) THE GOLD STANDARD! Durable CRs Must balance Toxicity with magnitude of the benefit Improvement of tumor related symptoms
Prolonged time to recurrence or Disease Free survival Palliation : Objective response plus improvement of symptoms Response : hormonal therapy

SURVIVAL IS THE GOLD STANDARD FOR CLINICAL BENEFIT Time from randomization to death (intent to-treat-analysis of all randomized) Basis for traditional approval of all of the following: Taxol and cisplatin: first line Rx of ovarian Ca Gemzar and cisplatin: first line Rx of lung Ca Camptosar: for first and second line therapy of CRC Taxotere: for treatment of metastatic breast cancer after prior chemo failure.

ACCELERATED APPROVAL
Serious or life threatening disease

Drug /Treatment provides benefit over available therapy Surrogate Endpoint of benefit, ratehr than Gold Standard survival Phase IV trial is mandated

SURROGATE ENDPOINTS
Less Toxicity than Standard of Care

Higher incidence of response (CR, PR); hematologic maintenance (e.g., MDS); cytogenetic response (CML); reduction of polyps (familial adenomatous polyposis); duration of response Quality of Life (abatement of tumor related symptoms {pain}, improved functional status, eliminate need for venous or central venous access Require another study confirming clinical benefit---should be ongoing at time of approval

EFFICACY AND SAFETY OF A SPECIFIC INHIBITOR OF THE BCR-ABL TYROSINE KINASE IN CHRONIC MYELOID LEUKEMIA
BRIAN J.DRUKER,M.D.,MOSHE TALPAZ,M.D.,DEBRA J.RESTA,R.N.,BIN PENG,PH.D., ELISABETH BUCHDUNGER,PH.D.,JOHN M.FORD,M.D.,NICHOLAS B.LYDON,PH.D.,HAGOP KANTARJIAN,M.D., RENAUD CAPDEVILLE,M.D.,SAYURI OHNO-JONES,B.S.,AND CHARLES L.SAWYERS,M.D.

White Cell Count

(cells x 10-3 / mm3)
100

Ph Chromosome + Cells
100

% in Metaphase
0 30 60 90 120 150

80 60 40 20 0 0 100 200 300 400 Duration of Treatment with STI571 (Days)

10

Duration of Treatment with STI571 (Days)

NEJM 344: 1031, 2001

Major Cytogenetic Response (%)

RESPONSE FOR IMATINIB VS. INTERFERON AND LOW-DOSE CYTARABINE IN LOW100 CHRONIC-PHASE CML CHRONIC80 60 40 20 0 0 3 Combination therapy p<0.001 6 9 12 15 18 21 Months after Randomization 24 27 Imatinib

Druker et al, NEJM 348: 994, 2003

ETHICS OF CLINCIAL TRIALS: FUNDAMENTAL POINTS

Clinical studies in oncology are performed in a human population which is burdened with cancer, and usually involve risk To be defensible on ethical grounds, a clinical trial must have significant scientific value, and must be conducted properly. Useless trials are inherently unethical Useful trials are unethical if appropriate procedures are not followed

WHAT IS RESEARCH?
Research = systematic investigation designed to develop or contribute to generalizeable knowledge (CFR 46.102 (d)) Clinical Research: Research involving human subjects. Human subject: living individual about whom an investigator conducting research obtains: data through intervention or interaction with the individual, or identifiable private information (CFR 46.102 (f)).

ETHICS OF CLINICAL TRIALS: FUNDAMENTAL POINTS

Clinical studies in oncology are performed in a human population burdened by cancer, and usually involve risk. To be defensible on ethical grounds, a clinical trial must have significant clinical value and must be conducted properly Useless trials are inherently unethical Useful trials are unethical if appropriate procedures are not followed

ETHICS OF CLINICAL TRIALS: DECLARATION OF HELSINKI-1 HELSINKI Follow a series of evolutions in modern concepts of proper clinical research Nuremburg Code : after Nazi doctors crimes Belmont Report Ethical clinical studies are scientifically sound Ethical clinical studies require a formal written protocol reviewed by an independent committee = Institutional Review Board (IRB) Research led by scientifically and medically qualified people

ETHICS OF CLINICAL TRIALS: DECLARATION OF HELSINKI-2 HELSINKI The importance of the research objective should be in proportion to the risk to the subjects Concern for subject well being supersedes the interests of science and society Privacy, integrity , physical, mental, and emotional health of subjects protected to greatest extent possible Physicians should abstain from research unless they are satisfied with the potential hazards in relation to the knowledge to be gained

ETHICS OF CLINICAL TRIALS: DECLARATION OF HELSINKI-3 HELSINKI Results of human experiments must be published in a timely manner; unpublished clinical studies are inherently unethical. Freely given informed consent must be obtained from all subjects following adequate explanation of the aims, methods, anticipated benefits, and potential hazards and discomforts Physicians must not take advantage of subjects dependent relationship (employee, excessive payment e.g.)

ETHICS OF CLINICAL TRIALS: DECLARATION OF HELSINKI-4 HELSINKI In the case of legal incompetence, informed consent must be obtained from a legal guardian In the case of minors, the minors assent should be obtained in addition to the consent of the guardian Permissible for verbal description for the visually impaired; not permissible to perform interpretation of English informed consent to a non-English speaking patient.

THE U.S. COMMON RULERULE1

Statutory definition of elements of informed consent State that the procedure is research; Purpose/duration/procedures to be followed; identification of the research procedure Description of reasonably foreseeable risks Description of likely benefits or lack thereof Disclosure of alternative procedures or treatments

THE U.S. COMMON RULERULE2

Statement of the degree of confidentiality ( who the research results will be available to) An explanation as to whether compensation or medical treatments are available in the event of injury Contact information re research questions, research subjects rights, and in the event injury from the research is perceived by the patient A statement that the research participation is voluntary Refusal to participate incurs no penalty or loss Subject free to withdraw at any time

SUMMARY
Oncology drug development is in an exciting time: New targets New molecules Better Understanding of Disease Life Scientists are the "gate keepers" for interesting opportunities for drug discovery and development A laboratory observation is conceivably a starting point for an train of investigations with scientific, clinical, ethical, and societal implications