Modification of Radiation Response

Biological Modification

 repair of radiation damage

 dose rate effects

 fractionation

Repair of DNA damage Three types of dam a ge

 Lethal d a mage- irrepairable, l eads to cel l death

 Potentially lethal damage

 Sublethal dam a ge

Potentially lethal damage (PLD): com ponent of radia tion damage that can be m odified by post-irra diation conditi ons.

PLD repair has been dem onstrated in vitro.

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Survival increases when:

 Cells are p r evented from dividing for ≥ 6 hrs.

 Cells are k e pt in saline or plateau (G 0 ) phase for ≥ 6 hrs.

PLD repair has been dem onstrated in vivo : relevant to the clinical situation.

 Survival increases when a del a y is intr oduced after irradiation and before the excision assay.

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 in vivo/in vitro assay

 smaller tum o rs well oxygenated

PLD repair is signi ficant for X rays

PLD rep a ir does not occur with neut rons (or other high-LET radiation).

Sublethal Damage Repair : increase in survival observed if a given radiation dose is split into two fractions separated by a time interval.

Split dose experiments

24 o C incubation allows repair but prevents cells from p r ogressing through the cell cycl e.

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Changing the tim e in terval between doses illustrates sev e ral im portant radiobiological principles.

Split dose experimen t at 37 o C Chinese h a mster cell s

 Initial repair

 Partially synchronized survivors (S phase)

 6 hours later populati on is in a radiosensit i ve phase of the cell cycle (G 2 /M)

Three processes ar e o p erative: The three “R”s of radiobi ology

 Repair of sublethal damage

 Reassortment by progression of survivor s through the cell cycle

 Repopulation if interval between fr actions > cell cycle ti me

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Split Dose Summary

 Split doses increase survival, the shoulder of the curve is repeated with each dose.

 Extent of SLD repair correlates w ith the size of the shoul der.

 β com ponent causes survi v al curve to be nd and results in the sparing effect.

 Large shoulders indi cate a small α / β ratio.

 The dip between fractions (reassortm e nt) is only observed with fast-cycling cells.

High-LET radiation and SLD repair

 Little or no shoulder

 Little or no repair

 Little or no SLD rep a ir when split doses are given

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Clinical Implications of SLD repair:

If:

Early respondi ng tissues: SLD ~ 20% of total dam a ge Late responding tissues: SLD ~ 50% of total damage

Repair of SLD: t 1/2 ~ 0.5 – 1.5 hrs

“fading time” ti me for dama ge to be ~ 95% repaired

3-4 hrs i n early respondi ng tissues: SLD ~ 20% of total dam a ge 5-6 hrs i n l a te responding tissu es: SLD ~ 50% of tot a l damage

Fraction spacing should be > 6 hours to allow normal tissues to fully r e pair SLD.

The Dose Rate Effect

 For low-LET r a diation, dose rate is one o f the principle factors that determ ine the biologi c response.

 Low dose rate sparing results from re pair of SLD during the protracted exposure.

Idealiz e d fractionation experiment

Multiple small fractions approximate a continuous low dose rate exposure.

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Continuous low dose rate com p osite (dashed line) has no shoulder.

Chinese h a mster cell s

 60 Co exposure at various dose rates

 Most dramatic dose r a te effect is observed between 0.01 and 1 Gy/m in.

 Dose rate sparing va ries with cell type.

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Dose rate effect in vivo

 Intestinal epithelium crypt cell s in the mouse.

 Cell proliferation begins to domin ate at t h e lowest dose rate (0.57 rad/min).

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Do se ra te e f fe ct summa r y

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 Acute doses: shoulder signi ficant.

 Low dose rate: D 0 increas es, shoul der decreases.

 Redistribution: in some cell lines, a G 2 block results in increased sensitivity “ inverse dose rate effect”.

 Below critical dose r a te: G 2 block can be overcome, proliferation increases.

Modification of Radiation Response

Chemical Modification: radiosen sitiz ers and radioprotectors

Oxygen – best known and m o st general radiosensitizer

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 The slopes of survival curves for cells exposed to sparsely ionizing ra diation in hypoxia and in well oxygenated environm ents differ by about a factor of 3 – the oxygen effect.

 Hypoxia means low oxygen

 Anoxia means no oxygen.

Mechanism

 Mechanism ( s) of the oxygen effect rea lly not know n, 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 “fixa tion” of dam a ge in the lethal form and occurs in com p etition with chem ical repa ir of damage, perhaps by H atom donation from thiols.

Oxygen Enhancement Ratio (OER)

 OER = dose(hypoxi a)/dose(oxygenated) for the same biological effect .

 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).

 OER decreases as LET incr eas es: i.e., fraction of ra diation damage from direct action increases .

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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 radiosensitivity vs. oxygen concentrat ion.

 Half- m axi m al effect of oxygen at about 0.5% (3 mm Hg) O 2 .

 For com p arison, venous pO 2 is about 50 mm Hg an d arterial i s about 100 mm Hg; air is 155 mm Hg.

Import ance of the oxygen eff e ct

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

 Small tumors (<160  m) – no necrosis

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

 Sheath of growing cells always 100-180  m

 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-limited – as described by Thom linson and Gray (chronic hypoxia)

 Perfusion-limited – cells interm ittently hypoxic only when the blood flow transiently stops on their vessel (acute hypoxia).

Do hypoxic cells rea lly exist in tumors?

The first dem onstration of hypoxic cells in an experi mental animal tu mor was by Powers and Tolmach using the diluti on assay technique.

A two-com ponent survival curve was observed:

 Low doses: D 0 = 1.1 Gy (norm a l)

 High doses: D 0 = 2.6 Gy

 The ratio of about 2. 5 between the two D 0 values suggested the OER: the low dose com ponent was from oxyge nated cells and the high dose com ponent from hypoxic cells.

 Back-extrapolation of the high dose co m ponent to the y-axis gives the % hypoxic cells in the tum o r.

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Paired su rvival curves

 Steepest: fully oxygenated

 Shallowest: fully hypoxic

 Interm ediate curve reflects the actual m i xture and is biphasic.

 Asphyxiat e animal with N 2 several m i nutes before irradiation and subsequent in vitro assay.

 Air breathing

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Evidence for the presence of hypoxic cells in human tumors:

 Tum o r histology

 Oxygen electrode measurements

 Clinical gains with hyperbaric oxygen

 Studies showing anem ia is poor prognost i c factor also associated with local failure, but pre- transfusi o n help.

Reoxygenation

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First indications came after observations of 15% hypoxic cells in experimental tu mors after a variety of different radiation doses and fractionation schemes.

Im plication was that the tum o r cell population was dynam i c and able to readjust : reoxygenate.

A single 10 Gy dose kills virtually all oxic cells.

Tim e course of reoxygenation varies am ong different tumors.

Mechanism:

Vessel re-opening at short tim es. Neovascularization at long tim es.

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.

Halogenated pyrimidine s, BUdR and IudR

 Are incorporated into DNA in p l ace of thymin e. Th erefore, the tum o r cells must be cy cling faster than th e nearby dose lim iting norm a l tissues.

 IUdR and BUdR have si m ilar sensitization with X-rays, but IUdR is preferable clinically because it sensiti zes cells mu ch less to fluorescent light, so less harm ful side effects.

 Sensitize both hypoxi c and oxygenated cells.

 The degree of sensitization depe nds on the amount of halogenated pyrim idine 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 external beam therapy.

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Hyp oxi c cell radios ensitizers

Electron-affinic compounds tha t selectiv ely sensitize hypoxic cells while having 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

Sensitization enhancem ent ratio (SER) = D 0 (without sensitizer)/D 0 (with sensitizer)

Nitroimidazole class of com pounds m o st studied.

 Metronidazole (flagyl – a 5-nitroim i dazole) gave in vitro SER = 1.7 and in vivo SER = 1.3.

 Misonidazole (Ro-07-0582 – 2 – nitr oim i dazole) better sensitizer, with in vitro SER = 2.5 for hypoxic cells (no e ffect on oxygenated cells) and tumor SER up to 1.8.

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

Drugs that are preferentially toxic to hypoxic cells.

SR4233 (ti r apazamine)

Hypoxi c Cel l Cytotoxicit y Ratio ** Drug dose required to kill given proportion of aerobic cells divided by that needed to kill same fraction of hypoxic cells

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Killing hypoxic cells m a y have greater ther apeutic advantage than radiosensitizing 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 tumo r can be greater than if the tum o r is fully oxygenated. This occurs wh en the drug kil l s at least 50% of the hypoxic cells each ti me it is gi ven.

Results of clinical trials have been disappointing.

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

Possible reasons for failure of m i soni 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 l d 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 i ght be expected to give ER in hypoxic cells of about 1.3-1.4, could only give m i sonidazole with 6 fractions.)

Radioprotectors

Agents which decrease the response of cells to radiation.

Best radio p rotectors are thiols

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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

WR2721 (amifosti n e) and related compounds

“Covering” the SH with a phosphate group decreased the toxicity

In 1969, Yuhas and collea gues reported that WR2721 coul d protect normal tissues with less protection of tum o rs.

Note that the degree of protecti on depends on the normal 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 phospha tase in plasma mem b rane

 pH

 Oxygen concentration

 Endogenous thiol le vel

 Fractionation pattern (single ver sus 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, interest in WR2721 and it s 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.