Chemical Modifi cation of Radi ati o n Resp onse

Oxygen

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Oxygen – best known and m o st general radiosensiti zer

 The slopes of survi v al curves for cells exposed to sparsely ioni zing radiation in hypoxia and in well oxygenated environments differ by about a factor of 3 – the oxygen effect.

 Hypoxia means low oxygen, anoxia means no oxygen.

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Oxygen Enhancement Ratio (OER)

o D 0 (hypoxi a )/D 0 (oxygenated)

 = dose(hypoxia)/dose(oxygenated) for the same effect

 If the survival curves in both air and hypoxia extrapolate back to the same n value, the curves are said to be purely dose m odifying.

 OER is usually about 3 at high radiation doses, but often has a lower value of about 2 at low doses (at or below 2 Gy).

How mu ch O 2 is required?

Survival curves show that relatively little O 2 is need ed, e.g., as little as 1 00 ppm O 2 (0.075 m m Hg) causes significant sensitization com p ared to the response in anoxia.

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“K-curve” – plot of relative radiosensiti vi ty vs. oxygen concentr ation – shows half- maximal effect of oxygen at about 0.5% (3 mm Hg) O 2 . (For com p arison, venous p O 2 is about 50 mm Hg and arterial is about 100 mm Hg; air is 155 Hg.)

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Timescal e – Wh en must O 2 be present?

 Very fast response techniques show that O 2 m u st be present during irradiation or added within m illiseconds after irradiation in order to be sensitizing.

 For m o st practical purposes, it m u st be present during the radiation exposure.

Mechanism

 Mechanism ( s) of the oxygen effect r eally not know, although, clearly, O 2 acts at the free radical level.

 The reacti ons involved may be:

O 2 + e - aq  O 2  - or O 2 + R   R O 2 

 The later reaction is sometim e s called “f ixation” of dam a ge in the lethal form and occurs in com p etition with ch em ical repair of damage, perhap s by H atom donation from thiols (t o be discussed m o re below).

Import ance of the oxygen eff e ct

 Thom linson and Gray (1955) st udied sections of bronchial carcinom a

 Small tumors (<160  ) – no necrosis

 Tum o rs over 200  - necrotic centers surrounde d by sheath of healthy cells

 Sheath of growing cells always 100-180 

 They also cal culated O 2 diffusi on in tissues and found that all O 2 should be metabolized at a distance of 150-200  from a capillary , in good agreement with the observations of necrosis.

 Actually, there will b e an O 2 concentration gradient through the tu m o r, so some tumor cells will have enough O 2 to grow but will be radiobiologically hypoxic (and therefore radioresistant ). These cells may lim it the effectiveness of radiation therapy of tum o rs.

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This m odel is really a gross oversim plification of tum o r oxygenati on, but emphasizes the im portance of oxygen in radiation t h erapy and explains why a great deal of resear ch has been conducted into ways to overcome hypoxic cells.

Hypoxic cells may be of tw o types: Diffusion-lim ited – as described by Thom linson and Gray (chronic hypoxia)

Perfusion-lim ited – ce lls intermittently hypoxic only when the blood fl ow transiently stops on their vessel (acute hypoxia).

Dealing with the different types of hypoxia may require different methods.

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Do hypoxic cells rea lly exist in tumors?

The first dem onstration that they do exist in an experimental anim al tum o r was made by Powers and Tolmach using the diluti on assay technique. They observed a two com p onent survival curve:

Low doses – D 0 = 1.1 Gy – norm a l

high doses - D 0 = 2.6 Gy

The ratio of about 2. 5 between to two D 0 values suggested the OER: the low dose com ponent was from oxygenated cells an d the high dose com ponent from hypoxic cells.

Back-extrapolation of the high dose com pone nt to the y-axis gives the % hypoxic cells in the tum o r.

[insert hall 6.10]

Hyperbaric Oxygen (HBO)

Short ly after the identification of hypoxia as a potential cause of tum o r radioresist a nce, clinical trials were begun with hyperbaric oxygen.

Most trials have been relatively sm all and used unconven tional fractionation patterns, but in several there was to be an advantage, albeit s m all, to the use of HBO.

Problem s included:

- Questions of whether increas es in dissolved O 2 in plasma really resulted in increases in hypoxic tum o r cells.

- Some normal tissues m a y be of sufficient ly low O 2 to be sensitized by the HBO.

- Practical problem s such as patien t convul sions due to oxygen, patient com p lications in lungs and ear s, claustrophobia, danger of fi re and explosion, etc.

Use of carbogen (95% O 2 /5% C O 2 at 1 atm ) with or without perfluorochem icals may give as good or better results than HBO.

Evidence for the presence of hypoxic cells in human tum o rs: Tum or histology

Oxygen electrode measurements

Clinical gains with hyperbaric oxygen

Studies showing anem ia is poor prognost i c fa ctor also associated with local failure, but pre-tra n sfusion help.

Radiation Sensitizers

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Radiosensitizers

 Agents which enhance the response of cells to radiation.

 Ideally, radiosensit i zers would selectively sensitize tum o r cells while having no effect on norm al tissues.

Non-hypoxic cell radiosensitiz e rs

 Halogenated pyrim idin es, BUdR and IUdR

 Are incorporated into DNa in pl ace of t hym in e. Therefore, the tum o r cells m u st be cycling faster than the nearby dose0li m iting normal tissues.

 IUdR and BUdR have sim ila r sensitization with X-rays, but IUdR is preferable clinically because it sensitizes cells mu ch less to fluorescent light, so less harm ful side effects.

 Sensitize both hypoxi c and oxygenated cells.

 The degree of sensitization depends on the amount of halogenated pyrimidine incorporated into a cell.

 Clinical trials with IUdR are u nderw ay with gliomas and sarco m as with encouraging early results. The agent is being delivered with brachytherapy as well as ext e rnal beam therapy.

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Hyp oxi c cell radios ensitizers

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Hypoxic cell radiosensitizers – electron-affinic compounds tha t selectively sensitize hypoxic cells while havi ng no effect on oxygenated cells.

Ideal properties of sensitizer:

 Selectively sensitize hypoxic cells

 Chemi call y stable an d slowly metabolized

 Highly sol uble in water and lipids so can diffuse to hypoxic tum o r cells

 Effective t h roughout cell cycle

 Effective at low daily doses of radiation

SER = D 0 (without sensitizer)/D 0 (with sensitizer) Nitroim i dazole class of com pounds m o st studied.

- Metronidazole (flagyl – a 5-nit r oim i dazole) gave in vitro SER = 1.7 and i n vivo

ER = 1.3.

- Misonida zole (Ro-07-0582 – 2 – nitr oim i dazole) better sensitizer, with in vitro SER = 2.5 for hypoxic cells (no effect on oxygenated cells) and tum o r SER up to 1.8.

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Hyp oxi c cell cyt o t oxins; Bi oreducti ve agent s

(quinines, nitro com pounds, benzotriazine di-N-oxi des)

- Drugs that are preferentia lly toxic to hypoxic cells.

Agent	Hypoxi c Cel l Cytotoxicity Ratio *
Mitomyci n C	2
E O 9	5
M e t r o n i d a z o l e	2
M i s o n i d a z o l e	1 1
N i t r o f u r a z o n e	8 . 5
RSU 1069	67
SR4233 (ti r apazam ine)	50

* Drug dose required to k ill given proportion of aerobi c cells divided by that needed to kill same fraction of hypoxic cells

Data all fo r V79 cells; HCR values vary with cell line

 Killing hypoxic cells m a y have great er therapeutic advantage than radiosensit i zing them because:

 hypoxic cytotoxins kill cells resistant to radiation and m o st chem otherapy, producing com p lementary cytot oxicity.

 random fluctuations in acute hypoxia could create a situation where hypoxia could be used to advantage.

Modeling studies show that if a hypoxic cytotoxi n is given with every dose fraction, the overall kill in a hypoxic tumor can be greater than if the tum o r is fully oxygenated. This occurs when the drug ki lls at least 50% of the hypoxic cells each time it is g i ven.

However, for a hypoxic cytotoxin to be e ffective in a fractionated regim e n, there m u st be rehypoxification between fractions.

Results of clinical trials have been disappointi ng.

Table 1. Results of random ized controlle d trials of radiosensit izing methods

	Hyperbaric Oxygen	Metronidazole	Misonidazole
Therapeuti c Benefit	3	0	2
Signi ficantly Im proved Results	0	2	4
Margin in favor (not signi ficant)	6	0	2
No Difference	6	4	30
Margin Against (not significant)	0	0	1
Adverse Response	0	0	0
	15	6	39

(from Dische in Malaise et al 1989)

Possible reasons for failure of m isoni dazole in clinical trials include:

- Rel atively small sam p le size an d heter ogeneous population m a y have precluded observation of a sm all effect.

- SER may be lower at clin ically relevant d o ses.

- Misonidazole has cum u lative neuropathy, which limited the dose that cou ld be given and the num ber of fractions with which it could be given. (Total of 12 g/m 2 , so with single dose of 2 g/m 2 , which m ight be expected to give ER in hypoxic cells of about 1.3-1.4, could only give m isonidazole with 6 fractions.)

Radioprotectors

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Radioprot ectors – agents which decrease the response of cells to radiation. Best radio p rotectors are thiols

Dose Reduction Factor (DFR) = Protection Factor (PF) =

dos e o f radiatio n i n t h e presenc e o f th e drug dose of radiation in t h e absence of the drug

to produce a given level of effect.

Proposed mechanism s for radioprotection by thiols: TH + · OH  T · + H 2 O (TH = target)

RSH + · OH  RS · + H 2 O 2 chemi cal p r otection

The rat e of the scavenging reaction is i ndependent of the presence or absence of oxygen, so scavenging will not explain th e differential protection by thiols in hypoxia and air.

Donation of H ato m s to organic radicals, in co m p etition with damage “fixation” of those radicals by oxygen

T · +RSH  TH + RS · chemi cal r e pair

T · + O 2  TO 2  dam a ge fixation Consum ption of oxygen, so hypoxia is produced

2RSH + O2  RSS R + H 2 O 2

WR2721 (am i fostime) and related com pounds

Covering the SH with a phosphat e group decreased the toxicity

I n 1969 Yuhas and colleagues reported that WR2721 could prot ect norm a l tissues

with less protection of tum o rs.

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Note that the degree of protecti on depends on the norm a l tissue type.

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However, radioprotection by WR2721 is not restri cted to normal tissues; tum o rs can be protected.

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Som e factors which affect the degree of radioprotection of normal tissue or tum o r by WR2721 include:

 Rate and extent of uptake of WR2721, which depends on:

 Concentration of alkaline phosphata se in plasma mem b rane pH

 Oxygen concentration

 Endogenous thiol le vel

 Fractionation pattern (single versus m u ltiple doses)

Clinical trials have shown side effects such as hypot ension, nausea, vom i ting, and hypocalcem i a. Hypotension has b een the dose limiting toxicity.

 Only a few clinical trials with radiati on have been undertaken. Although acute reactions to radiation appear to be protected, the effects on lat e radiation reactions remain to be evaluated in long t e rm studies. Acute reactions do not always predict late r e actions, so dos e escal a tion must proceed cautiously.

 Clinical trials suggest am ifostine may be useful to protect against nephro-neuro- and oto-toxicity from cis -platin treatment and cyclophospha m i de-induced granulocyt openia.

 More recently, interested in WR2721 and its derivatives has centered on observations that these drugs effectivel y protect against radiation-induced and some drug-induced m u tations and neoplastic transform a tion.

Protecto r	Treat ment	Endpoint	PF
WR-1065	Gamma rays	HGPRT mutations	5.1
WR-1065	Neutrons	HGPRT mutations	3.3
WR-1065	cis -PT	HGPRT mutations	7.1
WR-1065	HN 2	HGPRT mutations	3.4
WR-1065	BLM	HGPRT mutations	2.8
WR-1065	gamma rays	Transformation	6.0
WR-2721	gamma rays	Preneoplastic lesions	9.7
WR-2721	gamma rays	Tum o r induction	3.1
Mixture	X- rays	Tum o r induction	1.4

F r o m G r d i n a et al

REFERENCES:

E.J. Hall, Radiobiol og y fo r th e Radiologist , 5 th ed., Lippincott, Philadelphia, Chapters 6, 9 and 25.

E . P. M a la ise , M. G u ic ha rd and D.W. Siemann, Eds., Chem ica l Modifier s of Cancer Treat ment , Pergam on Press, NY, 1989.

T. W a sser m an, D. Siemann and P. Workman, Eds., Chemica l M odifier s o f Cancer Treat ment , Pergam on Press, NY, 1992.

H. Barteli nk and J. Overgaard , Eds. Radiotherapy and Oncology , Vol. 25, Suppl. 1, 1991.

C.N. Coleman, Radiatio n an d Chem otherap y Sens itizer s an d Protector s i n Cancer Chem otherap y an d Biotherapy , 2 nd edition, Chabner and Longo, Eds., 553-584, 1996.

J.M. Brown, The Hypoxic Cell: A Target for Selective Cancer therapy – Eighteenth Bruce F. Cain M e m o rial Awar d Lecture, Cancer Research , 59, 5863- 5870, 1999.

M. Werner-Wasik, Future Developments of Am ifost ine as a Radioprotectant,

Seminars in Oncology , 26, 129-134, 1999.