CA1269658A - Method and materials for sensitizing neoplastic tissue to radiation - Google Patents

Abstract

ABSTRACT

Tumours are sensitized to radiation by administration of 5-chlorodeoxycytidine (5-CldC) or 5-halo-2'-halo-2'-deoxycyti-dine or uridine derivatives. Tetrahydrouridine (H4U) and/or 2'-deoxytetrahydrouridine (dH4U) is preferably coadministered with the deoxycytidine derivatives to inhibit deamination of the deoxycytidine derivatives. Optional prior concurrent treatment with agents to reduce the amount of competing metabolites to favor CldC , such as 5-fluorodeoxyuridne, results in a procedure that significantly increases the dose effects of X-radiation.
Pharmaceutical compositions suitable for the sensitization of tumors to radiation are also disclosed, as are novel compounds.

Description

The present invention per~ains to combination radiotherapy of tumors and more specifically to pharmaceutical compositions and methods of treatment and therapy designed to sensitize tumors in animals, notably humans, and render them more sensitive to radia~lon, thus signi~icantly reducing the amount of r~diation required tG kill neoplastic cells while at the same time making the radiation far more tissue specific to the tumor site.
More conventional radiation sensitizers are hypoxic 1~ cell sensitizers such as the antifilarial agent, misonidazole, which causes the complexities of neurotoxicity when it is utilized in humans. The 5-halogenated pyrimidine analogs are very distinct agents from the hypoxic cell sensitizers, for they have a completely different mode of action. We have found that 1~ cultured mammalian cells, when exposed to bromo-2'-deoxyuridine (BrdU) or other halogenated analogs of thymidine incorporate the compound into DNA resulting in sensitization of these cells to x-irradiation. Rapid catabolism or degradation of BrdU has limited its clinical e~fectiveness for the sensitization of rapidly 2a growing neoplasias.
We have determined that 5-chloro-2'-deoxyuridine (CldC), which is not readily degraded due to the 4-amino group that protects the compound from catabolism by nucleoside phosphorylases, is anabolized by a different set of enzymes than the corresponding dU analog. In addition CldC is less cytotoxic than CldU. An ob~ect of the present invention ls to define a class of stable, selactive cell sensitizers that are not easily catabolizsd against tumor, both rapidly and moderately growing and malignant, and that will allow the X-ray therapist to focus 3~ the X-ray beam (or other source of radiation) on the tumor tissue site using significantly less, say one-fourth, of the dose of radiation otherwise requ~red to achieve the same extent of tumor kill without damage to the underlying tissue. Expressed in X~
~a .

~nother way, the radiotherapist is able to more aggressively kill neoplastic tissue while causing no more damage to normal tissue using the procedures and ma~erials o~ the presen-t invention than with conventional modalitiss of irradiation.
Thus an ob~ect of the present invention is to provide therapeutic materials and procedures for treating skin lesions using, for instance, ultraviolet llght, near visible light (313 nm), and for solid tumors: X- or gamma ray, beta, neutron and other radiation entities.
Another obJeGt of the invention is to sensitize possible sites of metastic invasion to radia-tion, particularly x-ray, using the disclosed materials and procedures.
Should the patient develop ~oxicity ~rom the mildly aggressive therapy employed, particularly with the pretreatment l~ with FdU and PALA either before or with the sensitizing composition, thymidine or deoxycytidine, two non-toxic metabolites~ may be provided the patient at ~he conclusion of radiation therapy. These serve to mitigate the untoward effects, if any, of the drug therapy by antagonizing or reversing any ~o toxic effects of the chemotherapeutic agents employed.
Tha method of the present invention may be used primarily with X-ray or ~amma (derived from cobalt, for example) radiation. We also consider the procedure to be effective with the use of ultraviolet light, near visible light (313 nm) for skin lesions and for beta, neutron and othar radiation entities.
Tha molecular basis of sensitization has been clearly established for ultraviolet light (260 nm) and near visible light 3a 3~

(313 nm~ (hoth nonpenetrating) and for X- or qa~ma-raaia~ion.

SU~RY OF T~E INVENTION

According to one aspect, patients having tumors S requiring radiation therapy zre aaministered, preferably on a slow release basis, 5-chloro-2-deoxycytidine and/or 5-chloro 2'-halo-2'-deoxycytidine, wherein halo is fluoro, chloro, bromo or iodo, preferably chloro. The deoxycytidine compound is preferably administered with a a deamination inhibitor, prelerably tetrahyarouridine ~H4U) and/or 2'-deoxytetranyarouridine (dH4U~ for a period of time until amounts sufficient to sensitize the t~mor tissue to radiation are present in the tumor tissue. Opti.~ally, .he Datient is siven a pretreatment regimen designed to lower the metabolites competing for CldC, or a special limited diet is used for the same purpose. Drug treatment is suspen~ed and raàiation therapy initiated at the aosage required to kill .he ` exposed tumor tissue while avoiding or reducing sianificant damase to the underlying .issue. If toxicity is encountered in the pretrea~ment sensiti-zation procedure, thymidine or deoxycytidine may be administered to the patient immediately following the radiation treatment to counteract toxicity without ~S affecting selective tumor kill.
Pharmaceutical compositions providing the re-quire~ amounts of 5C1-2'-halo'2'-dC or of Shalo-2'-halo

-2'-dU, as well as compositions providing the required amoun~s of 5-CldC and H4U or dH4U in pharmaceutically ,` acceptable formulations are also described.

~ he novel compounds forming one aspect of the present invention may be prepare~ according to methods stand~rd in the act as expounded in the literature references cited herein, particularly via methods similar to those disclose~ by Codington, Doerr and Fox, in J. Or~. Chem 29,55~ (1964~.

In general, one particular process may entail reacting a suitable 2,2'-anhydro nucleoside under conditions which halogenate the 2' position. For example, where a 2'-chloro substituent is desired, the 2,2'-anhydro nucleoside i9 reacted with anhydrous HCl by heating under pressure in suitable solvent e.g. dioxane to generate 2,2'-chloro-nucleoside. The 2' position may be substituted with other halogens such as fluoro, bromo and iodo using known reagents to replace the anhydrous HCl t~ith due alteration to specific reaction conditions.
To prepare a compound possessing a 5-halo substituent in addition to a 2'-halo substituent, the 2,2'-halo-nucleoside qenerated as described above is further reacted with a halo~enating agent. Halogenating aqents suitable for this purpose will be apparent to those skilled in the art, examples of which include N-chloro-succinimide, bromine gas and the like. The desired compound may be recovered from the reaction mixture using standard techniques.
According to another aspect, patlentS are administered with a 5-chloro,5-bromo or 5-iodo-2'-halo-2'-deoxyuridine compound, preferably the 5-bromo or 5-iodo ~ompound, ~herein halo is fluoro, chloro, bromo or iodo, ~, .

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prsferably chloro. In this instance, because the deoxyuridine moiety does not contain an amino substi~uent, it is not necessary to inhibit deamination thereof (this is discussed in more detail below), and so administration of H~U and/or dH~U with the 5 deoxyuridine compound is not required.
Rapid catabolism and generalized toxicity have limited the use of 5-haloger.ated analogs of deoxyuridine as tumor sensitizers. In one approach to this problem, 5-halogenated analogs of deoxycytidine (dC) or of 2'-halo 2'-deoxycytidine were utilized, which are not catabolized unless they are deaminated.
To prevent deamination by cytidine deaminase (CD), which is axtremely active in human serum, it is preferred, according to the invention, to administer tetrahyrouridine (H4U), a potent inhibitor of this enzyme, either concurrently with, or at about tha same time as, administration of the deoxycytidina compound.
Our pravious enzyme kinetic studies with 5-bromo-2'-daoxycytidine (BrdC) and 5-iodo-2'-deoxycytidine (IdC) indicate that they would not be suitable, in thi~ approach to circumvent catabolism, because they are poor substrates for deoxycytidine 2~ kinase. Unlike BrdC, chlorodeoxycytidine (CldC) does not rPquire deamination at the nucleoside level for its anabolism because it possesses a reasonable Km value (56 M) with respect to mammalian daoxycytidine kinase compared to 400 M for BrdC and 2 M for dC.
Studies with HEp-2 cells suggest that CldC (~H4Ut is metabolized as follows: CldC l>CldCMP ~ CldUMP 3~CldUTP 4-~DNA
~l=deoxycytidine Xinase, 2=deoxycytidylate deaminase (dCMPD), 3-thymidylate kinase, (4-DNA polymQrase). These four enzymes are elevated in many human tumors. For example, dCMPD activity in human malignant tumors is 20-80 fold higher than that of normal

3~ tissue.
In X-radiation studies with HEp-2 cells, we have X

obtained 3.4-3.7-fold does incre2se effects. Cells were-~eincu~a,ed ~ h nhlbitors of ce novo pyrimidine synthesis: N-(Phosphonacetyl)-L-2spartate (PALA) and 5-fluorodeoxyuridine (FdU) for 20 hours and 5 hours, S respectively and then incubated in the presence of 0.1 or O.Z mM CldC and H4U (100 ~) for 64 hours. These conditions resul~ in 40-50% substitution of CldU for thymidine in DNA. Viabilities of 10% + 4 to 12g ~ 5 were obtained for drug-treated unirradiated cells. In-hibitors of thymidylate synthetase that are more DNA
and tumor selective than FdU are also within the ambit of this invention. CldC and its metabolites are not toxic unless deamination occurs. CldC should be con-verted preferentially to CldUMP in tumors possessing lS hi~Jh levels of dC~PD and then be further anabolized to Clc~TP, resulting not only in radiosensitization but also in selective tumor toxicity, presumably as a result of inhibition of ribonucleoside diphosphate reductase by C'dVTP.
2`~ Addition of dH4U, which resulis in inhibition of CD and dCMPD, has enabled us to study radiosensitization due to incorporation of CldC 2S such into DNA. However, 2S no~ed above, it is not necessary to use dH4U (or H4U) with the 5-iodo or 5-bromo 2'-halo-d~ derivatives since deamination is not a problem in those derivatives.
The preferred materials used to carry out the ~resent invention, including abbreviations and structural formulae, are listed in Table I below.
5-chlorodeoxycytidine (CldC) was obtained from 3`~ Calbiochem-~ehring and has been described in the literature as an antiviral (antiherpetic); see Fox, Mekras, Bagwell and Greer et al, Capacity of Deoxycy-tidine to Selectively Antagonize Cyto~oxicity of 5-Ralogenated Analogs of Deoxycytidine Without Loss 3~ of Aniherpetic Activity, Antimicrob Agents Chemother., Vol. 22, No. 3, p. 431-441 (Se~t. 1982). Additionally-.

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DeClercq e~ al used CldC in cell culture studies (no indication is given for use in cancer therapy) the data indicating CldC was unre~arkable in the system employed; see ~Role of Deoxycytidine Kinase in the Inhiblting Activity of s-substi~uted 2'-Deoxycytidines an~ Cytosine Arabinosides on Tumor Cell Growth",J~ Balzarini, and DeClercq, Erik, Molecular Pharmacology, Vol.
23, p. 175-181 (198~).
The 5-chloro-2'-halo-2'-deoxycytidines as well as the 5-chloro, 5-bromo or 5-iodo~2'-halo-2'-deoxyuridine derivatives may be prepared according to the procedure described by Codington, Doerr and Fox, Nucleosides, XVIII Synthesis of 2'-Fluorothymidine, 2'-Fluorodeoxyuridine, and other 2' halogeno-2'-deoxy Nucleosides, J. Org. Chem., 29, 558 (1964).
Tetrahydrouridine (H4U) was obtained as a gift from the Drug Development Branch of the National Cancer Institu~e, Bethesda, Maryland, its synthesis is described by Hanze, Catalytic Reduction of Pyrimidine Nucleosides, J. Amer. Chem.
Soc., 8g 6720-6725 (1967). 2'-deoxytetrahydrouridine (dH~U) inhibits cytidine beaminase and when phosphorylated it also inhibits deoxycytidylate deaminase. The synthesis of H~ U and dH~H are described in U.S. 4,017,606 (Hanze et al). To our knowledge dH4U has~never been utilized in tumor thsrapy with an analog of deoxycytidine.
5-fluorodeoxyuridine (FdU) is a known antitumor agent available from Slgma Chemical Company, 5-fluorodeoxycytidine ~FdC), which has a greater selectivity against tumors, may also be used. FdC, not previously been used for tumor radiosensitization, may be prepared according to Fox, J.J., Wempen, I., and Duschinsky, R., Nucleosides of 5-Fluorocytosine.
3a Proc. of the 4th International Congress of Biochemistry 15 p. 6 (1958). For the procedure of the present invention it is co-administered wlth tetrahydrouridine.
N-(Phosphonacetyl)-L-aspartate (PALA) has been used alone and with 5-fluororuracil (5-FUra) as an antitumor agent but not to pretreat tumor cells in con~unction with radiation. PALA
was obtained from -the Drug Development Branch of the National Cancer Institute at Bethesda, Maryland.
The strategy of pretreatment is to inhibit the de novo pathway of pyrimidine biosynthesis.
The use of 5-trifluoromethyl-2'-deoxycytidine (F3methyldC) together with tetrahydrouridine (H4U) in the treatment of Herpes or Herpes-like viruses is described in U.S.

4,210,638 to Sheldon Greer.

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TABLE I
Name Abbreviation Structure tetrahydrouridine H4U
H~LC
H
~H OH
2'-deoxytetrahydrouridine dH~U

~ Uo~c ~ ~ H

5-chloro-2'-deoxycytidine 5-CldC ~N ~ ~
~0~ O~N ~1 5-fluoro-2'-deoxyuridineFdU ~F

Ho~C
~ H N~l 5-fluoro-2'-deoxycytidine FdC N ~ f HO ~C~

N-(Phosphonacetyl)-L-aspartate PALA UO U
O o C~

o~ c---c--o ~oo ~ 2~$i$~i~
g The abbreviations used include: CD, cytidine-deoxycytidine deaminase; CHO, Chinese hamster ovary cells; CldC, 5-chloro-2'deoxycytidine, CldCMP, 5-chloro-2'-deo~ycytidine-5'-monophosphate; CldUMP, 5-chloro-2' deoxyuridine-51-monophospha-te;
dC, deoxycytidine; dCK, deoxycytidine kinase; dCMPD, d~oxycytidyla~e deaminase; dH4U, 2'deoxytetrahydrouridine; dT, thymidine; dU, 2'-deo~yuridine; dUMP, 2'-deo~yuridine-5' monophosphate; FdC, 5-fluoro-2'-deo~ycytidine; FdU, 5-fluoro-2' deoxyuridine; FdUMP, 5-fluoro-2'-deoxyuridine-5'-monophospha-te;
F3methyldC, 5-trifluoromethyl-2'-deoxyoytidine; FUra, 5-fluorouracil, HEp-2, human epidermoid laryngeal carcinoma cells, H~U, tetrahydrouridine; TK, thymidine kinase; TS, thymidylate synthQtase; TTP, thymidine-5'-triphospha-te.
While not wishing to he bound by any theory we offer the following as a further explanation of the possible mode of action of CldC as a radiosensitizing agent when coad~inistered with H~U as well as with dH4U. With a low concentration of ~etrahydrouridine to protect the nucleoslde analogs from systematic catabolism, one may envision that BrdC and IdC as well ~o as CldC will act as selective radiosensitizers against tumors with high levels of cytidine deaminase. At higher concentrations of H~U, CldC should be converted preferentially at the tumor site to CldUMP in human tumors possessing high levels of deoxycytidine kinase and dCMP deaminase. When anabolized to CldUTP not only ~5 will there be selective tumor toxicity because of inhibition of ribonucleoside reductase by CldUTP, but in addition the incorporation of CldU into DNA will lead to tumor radiosensitization. Selectivity will result not only because of accelerated DNA synthesis, but because of elevation of key 3~ enzymes in the tumor that are critical for this strategy; in addition, there will be the customary selectivity that is associated with ~t~ ;D7'~

utili~ing a focussed beam of irradiation overlying the iumor. l-nis approach~is amena~ie to rescue with deoxycytidine and thymidine immediately after irradiation. A typical irradiation experiment in the S mouse would involve an i.p. injection of CldC + H4U every 8 to 10 hours in a 30 to 36 period, for example. The uoal is to obtain substantial incorporation of CldC into both strands of DNA. The use of dH4U may result in the incorporation of CldC as such into the DNA of cells.
10 However, in this case there may be less selectivity against the tumor for we are probably only exploiting the elevation of dC kinase that occurs in tumors.

EXAMPLES OF THE INVENTION

To cetermine whether 5-chlorodeoxycyt~dine (CldC) 15 and tetrahycrou~idine (H4U) has potenti21 as a radio-`sensitizing combinationj we tested these agents in HEp-2 cells. These cells were chosen as our mocel system since they possess elevated levels of activity of deoxycytidine Xinase (dCX), cytidine 2eaminase ICD) 20 and deoxycytidylate deaminase (dCMPD); the enzymatic profile necessary for metabolic conversion of CldC to an established radiosensitizer, CldUTP. TMP kinase and DNA polymerase are also elevated in tumors; these elevations should assure further preferential ~5 incorporation of CldU into tumor DNA.
Many of the e~periments described below were done utilizing only one dose of radiation (usually 500 or 600 rads). Although one can not accurately calculate dose- `
increase effects based on such limited data, we have 30 nonetheless pursued our studies in this manner in order to test many different combinations of metabolites and antimetabolites in the same experiment. Naturally, we are missing important features seen o~y at low doses.
~owever, this approach fulfilled the need to search and find optimal regimes. We have ~ound that sensitizing e~fects are more pronounced at doses o~ 500 or 600 rads. Preliminary experiments that demonstrate some of the sensitization effects that have been obtained are described below.
CldC (and H4U), our stora~e and DNA- and target-directed form of CldU, coadministered wi~h metho~rexate gave an enhancement ratio of 2.0-fold. This is illustrated in Figure 1.
This effect was obtained with a viability of 66%.
In the next series of e~periments, cells were pretreated prior to CldC~+ ~4U administration with the inhibitor of de novo pyrimidine biosynthesis, N-~Phosphonacetyl)-L-aspartate (PALA). PALA is a potent inhibitor of aspartate transcarbamylase and causes a depletion of intracellular pyrimidine pools. Liang et al found that PALA and fluorouracil lS (FUra) were synergistic when PALA was administered prior to FUra.
These investigators speculated that the reason for this synergistic interaction was due to marked decreases in dUMP pools in cells preincubated with PALA. Decreased dUMP pools should help potentiate FdUMP inhibition of thymidylate synthetase leading to decreased levels of TTP; which would then result in less competition for the incorporation of CldU into DNA and greater activity o~ dCMPD. This i~ the rationale for the use of the combination of PALA and FdU in radiation experiments.
Furthermore, PALA, by reducing intracellular pyrimidine ~S biosynthesis may lower competing substrates for the activation of CldC to CldUTP.
In recent experiments PALA has been administered 12-20 hours prior to FdU pretreatment, which is ~or 6 hours. Evans et al have shown that FdUMP pools persist after FdU or FUra 3d administration. Coexposure of HEp-2 cells to GldC + H4U and FdU
for 48 hours in comparison to pretreatment with FdU or 6 hours does not lead to a greater enhancement of radiosen~itization by CldC + H4U but only results in greater cytotoxicity. Following the pretreatment schedule described above, we have been able to 3~

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lowar the concentration o~ CldC (from O.6mM to O.2mM) and achieve better sensi~ization to X-ray. These results are illustrated in Figure 2. CldC at 0.2mM gives a dose increase effect of 3.6 with an associated 12.4% + 5.1% ~+S.E.) viability, whereas CldC at a concentration of 0.6mM results in a 3.0-fold dose increase.
In the experiment summarized in Figure 3 we attempted to lower the concentration of CldC ~urther, but lost sensitization at a concentration of O.05mM. In an effort to minimize the number of manipulations performed, cells were exposed to PALA and FdU for 21 hours as a single pre-trea-tment, but this resulted in a significant loss in viability (viability aquals 1.6~) with no gain in radiosensitization (a 1.9 dose-increase). A 3.8-fold dose increase e~fect was achieved with conditions similar to those of the previous experiment with 9.8%
lS + 4.0~ (+S.E.) viability.
The rationale for utilizing PALA and FdU pretreatment is to achieve greater radiosensitization with CldC + H4U; however our approach is strengthened by the fact that PALA and fluorinated pyrimidines are agents which are effective in combination chemotherapy. The conversion of CldCMP to CldUMP at the tumor site because of elevated levels of dCMP deaminase, is an example of ~umor-dlrected toxicity as is the case with FdC.
The target enzyme in CldC therapy is presumably nucleoside diphosphate reductase, which is likely inhibited by CldUTP. FdC
2S pretreatment rather than FdU is currently believed to achieve a greater measure of tumor- and DNA-directed toxicity with no loss of radiosensitization with CldC, H4U and PALA in animal systems.
The experiment summarized in Figure 4 illustrates our second approach with CldC; that is, to examine the 3a ~2~

radiation effects resulting from the incorporation of ~ldC as -uch (without prior deamination to-CidU) in DN~.
This may be accomplished by dH4U, which as dH4UMP in-hi~its both deoxycytidine and deoxycytidylate deaminases.
If both sites of deamination are blocked, the only anabolic route for CldCMP will be to become phosphory-lated further to CldCTP. In this initial experiment a 1.8-fold dose-increase effect was obtained with CldC
and dH4U.
In subsequent experiments we sought to lower com eting dCTP pools by utilizing 3-deazauridine.
Deazauridine is a potent inhibitor or CTP synthetase and did not appear to enhance sensitization in this experiment. In the bar graph depicted in Figure S a 2.0-fold dose-increase e fect was ob.ained by CldC, dH~U and deazauridine with 21% viability~
It should be noted that the most striking effects we have obtained have been with the use of CldC and H4U
rather than with CldC + dH4U. That is, the combination OI CldC + H4U has resulted in a 3~4 to 3.8 dose enhance-ment eflect with appropriate pretrea~ment (PALA and an F-pyrimidine analog).
Figure 6 summarizes an experiment in which a 3.4 dose enhancement effect is displayed wnen FdC + H4U
~S replaces FdU in the pretreatment procedure prior to the addition of CldC and H4U~ 5-fluorodeoxvcytidine +
tetrahvdrouridine should result in tumor directed toxicity; that is, it should be more tumor specific than FdU. Thus ~dC + H4U may be used to obtain greater efficacy without loss of radiosensitization.
Figure 7 summarizes an experiment in which we have demonstrated .hat lowering both PALA and CldC concen-trations resultsin significant loss of radiosensitization without a subs.antial decrease in toxicity. FdU and FdC + H~U pretreatment are essentially equivalent in terms of effective CldC radiosensi.ization, with FdC +

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H~U displaying less toxicity, whlch is desirable. A maximum 3.4 to 3.6 dose enhancement effect was displayed in this experiment.
BrdU has been shown under the most optimal condi-tions in cell culture to display a 3.5 to 4.0 doæe-increase effect with X-ray and with ultraviolet light. We have now achieved comparable results with a combination of agents that will lead to the circumvention of catabolism and to tumor selectivity -- two features not readily achieved with BrdU. Most importantly, with this method one may irradiate a tumor at 1/4 the dose to pr~vent damage to underlying tissue or to more aggressively irradiate a tumor without an increase in damage to normal tissue.
Pharmaceutical Presentation -- pharmaceutical compositions comprising, as the active ingredient(s), radiation-sensitizing effective amounts o~ 5-CldC and H4U and/or dH4U
1~ together with a pharmaceutically acceptable carrier or diluent, for intraperitoneal administration for animal studies, intraveneous, subcutaneous, intramuscular, oral or topical administration are included in the present lnvention. While the components of the composition may be administered separately, it is preferred to coadmlnister them as a mixture. The concentration of each of the active lngredients may vary from about 0.01 to abou~ 25~ by weight depending on the route of administration, the frequency of administration, the severity of the condition, the age, weigh~ and general physical condition of the patient being treated as well as the size and location of the tumor to be irradiated. Alternatively a more concentrated solutlon will be used, ~.g. 75gJ100 ml, or a slow i.v. infusion of a 0.1 to 25~ ~or higher) conc2ntration will be used. When the composition is in the form suitable for topical administration, 3~ or example a cream, th~ concentration of the total of 5-CldC and H~U or dH~U will generally vary from about 5 to 50 wt %, preferably about 5 to 20 wt.%, more preferably from about 5 to 10 wt ~ When the com-position is in the form suitable for intraperitoneal administration for animal studies, for example, an aqueous solution of CldC and H4U or dH4U the concentra-tion will generally ~ary from about 0.5 to 5% w/v, more usually from 1% w/v. For oral administration, the conceniration will generally be from 0.05 to 10 wt.~, pre-erably about 0.5 to 5 wt.%,and more preferably ~out 1 ~ 2Wt-%-hnen used ~or intravenous injec~ion, the concentra-tion of thP active components will vary from about 0.05 to about 5% w/v, preLerably about 0.1 to about 0.5~ w/v.
For in~ramuscular injection, the same concentratiOn as cescribed above for t~e intraDeritoneal mode or ad-ministration will be utili~ed.
Other me~ho2s of aaministr2.ion .,ay also be used.
Su~positories may be used for sustained release pur~oses.
Slow-release surgic21 implants are also envisage2.
The p'n2rmaceutically acceptable carriers or diluents employed in the compositions of the present invention may be any compatible non-toxîc material suited for mixing with the active compou~ds. When the com-position is in a ~orm suitable for ~arenteral use, Lor example intramuscularly or intravenously, the carrier ~5 which preferably is an aqueous vehicle, may also contain other conventional additives, such 25 a suspending agent ror example methyl cellulose or pol~vinylpyrrolidone (PVP), and a conventional surSact-ant. For oral a~ministration, the compositions can be " 30 formulated as aqueous solutions, suspensions, capsules or tablets, suitably containing appropriate carriers or `` diluents, for example lactose, starch and/or masnesium stearate for flavoring agents, syrups, sweeteners or coloring materials as customarily used in such pre-parations.
A preferred pharm"aceutical composition provices the patient with a total i.v. cos2ge of .rom 3 to 5 ml(cc~

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per dosage calculated on 70 kg body weight of the patient.
Clinical Protocol: The pa~ient will be given drugs i.v. or i.m. or in an oral or suppository form or in a slow release form. A slow release administration of CldC and tetrahydrouridine may be particularly advantageous. Different routes may be used for individual drugs in one treatment protocol.
PALA in 10 ml ampules containing PAL~ disodium (1.0 gram) with Edetate disodium (l mg) and NaOH to adjust pH to 6.5 l~ to 7.5 will be given to a cancer patient with a solid tumor at a range 2 mg to 300 mg/kg per dose preferably 5 to 20 mg/kg per dose and more likely 10 mg/kg per dose. Twelve to thirty-six hours later preferably 18 to 26 and most likely 24 hours later, FdU at a concentration of 10-75 mg/kg per dose, preferably 25 to 60 more likely 50 mg/kg would be adminlstered. Alternatively FdC
at simllar concentration range as FdU will be administered but in this case it will be coadministered with tetrahydrouridine at a concentration range from 10 to 200 mg/kg preferably 15 to 100 and more likely 25 mg/kg. The ratio of H4U to FdC will range from 4:1 to 0.2:1 preferably 1.5:1 ~o 0.75:1 more likely 1:1.
Three to 12 hours later preferably 4 to 8 hours, more likely 6 hours later the series of administration of 5-CldC and H~U will begin.
The dose of 5-CldC will range from 200 to 2500 mg/kg per dose, preferably 500 to 2000 mg/kg, more likely 1500 mg/kg.
The dose of H4U will be the same as that given with FdC
described above. The ratios are, however, different; namely, the ratio of H4U to CldC will range from 1:30 to 1:5, more usually 1:30 to 1:10 and more likely 1:15.
3~ This will be repeated at 6 to 18 hr. intervals more usually 8 to 12 more llkely 10 hr. intervals. The Deriod of reDeated CldC + H~U admi~is~ration will be 20 ~o 60 hrs, more usually 30 to 50 hrs more likely 34 to 48 hrs. ~sually CldC + H4U will be administered in 3 to 4 doses, 8 to 12 hrs apart.
` After tne last dose of CldC ~ H4U an interval of 4 to 8 hrs will ensue prior to irradiation. This period will more preferably be 6 to 14 hrs and more likely 8 to 10 hrs.
The interval between PALA/FdU or FdC antitumo~
therGpy and CldC/H4U sensitizer therapy, as well as the freouency o administration is determined by the skilled elimination drawing upon previous ex~eriences and ob-servations ~sing this regimen and therapy. The i~terval between drug therapy and radiation treatment may be varied as well T;~e r2diation dose, X-ray or ga,~a-ray, for example, will be either the same or 1/4 or 3/4 ~he dose given to pa~ients not receiving the pretreatment sensi-tization schedule. This will result in ei.he~ (a) ..ore aggressive tumor kill without increased damage to under-lying tissue when ~he 4/4 dose is used, or (b) equal tumor kill as that achieved in patients given no treat-ment but with much less damage to underlying tissues when tne 1/4 to 3/4 doses are used.
If toxicity is encountered due to the drug treat-ment schedule then thymidine at a ~ose of 50 to 750 mg/kg, more likely 100 to 500 mg/~g, preferably 200mg/kg will be given immediately after the radiation treatment.
This will be repeated 2 to 3 times at 8 to 12 hr inter-vals. This treatment is designed to counteract toxicity without adversely affecting selective .umor kill.
Deoxycytidine at a concentration range similar to thymidine can be given with or instead of thymidine.
When deoxycytidine is administered it will be given at a ratio of ~etrahydrouridine to deoxycytidine of 1:.05 to 1-5.*
This course of treatm~n~ can be repea~ed one week to two weeks later and repeated again until the patient receives a total dose of 3000 to 7000 rads. ~n this st~ategy, the patient may naed 1QSS total irradiation to achieve effective tumor kill.
This is one advan~age o~ the strategy. The dose of radiation which, in the past provided only partial remission, may result in long tPrm cures. That is the primary advantage of this approach.
The dosages and ranges are summar~zed in the following lo table:

TABLE~II
DOSABE**
Most A~ent General Preferred Preferred pretreating PALA 2-300 5-20 10 FdU 10-75 25-60 50 or FdC
~ +H4U*** 10-200 15-100 25 ~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Sensitizing CldC 200-2500 500-2000 1500 H4U ~ 10-~00 15-100 25 dH4U 10-200 25-125 50 2~ _ _ * Deoxycytidine with H4U or dH4U also be given 2-6 hrs. after each CldC treatment prior to irradiation.
** expressed in mg/kg body weight/dose *** when FdC ls utilized 3~ The a~ove dosages and ranges apply with respect to the 5-chloro-2'-halo-2'-deoxycytidine compounds, as well as the 5-chloxo-, 5-bromo- and 5-iodo-2'-halo-2'-deoxyuridins dsrivatives.
Toxiclty Stud~es: Our analysis of the potential of toxicity uslng the method herein disclosed indicates that, at most, a 5-~ weight loss was achieved with no deaths due to toxicity. This is viewed as a trivial and most tolerable weight loss considering the normal aggressive results of the administration of antitumor agents in radlation therapy.
The protocol employed was as follows:
Three animals were injected i.p. with PALA at a dose of 200 mg/k~. This was followed 24 hrs. later with an i.p. injection of 5-fluorodeoxyuridine (FdU) at a dose of 5 mg/kg. Four hrs. later they were given an i.p. in;ection of CldC (500 mg/kg) coadministered with 100 mg/kg te~rahydrouridine. The administration o~ CldC + H4U at the above indicated concentrations was repeated two more times at ten hour intervals.
Only 5~ weight loss occurred. No deaths occurred with this protocol.
lS This protocol was modified using 700 and 1400 mg of CldC/kg and a FdU concentration of 60 mg/kg and extensive incorporation of 5-chlorodeoxyuridi~e into DNA of tumor tissue was observed.
The procedures of our invPntion go beyond taking ~o advantage of the rapid growth of tumors. It exploits important quantitative differences in the levels of enzymes between neoplastic and normal tissue. CldUTP, a metabolité product of CldC when administered in the presence of H4U is preferentially formed in tumor tissue to result in tumor directed toxicity and ~5 radiosensitization. 8ecause the mode of radiosensitization of pyrimidine analogs differs from that of hyperthermy and hypoxid cell sensitizers, our procedure and strategy may be used with those modalities in radiation therapy.

3~

Claims (12)

WE CLAIM:

1. A pharmaceutical composition for sensitizing neoplastic tissue to radiation, comprising a radiation sensitizing amount of a compound of the formula:

(I) wherein A is -NH2 or =O, X is chloro, bromo, iodo or fluoro; Y is hydrogen, chloro, bromo, iodo or fluoro, and ? is a single or double bond, with the proviso that ? is a double bond when A is NH2 and ? is a single bond when A is =O, in association with a pharmaceutically acceptable carrier or diluent.

2. A pharmaceutical composition for sensitizing neoplastic tissue to radiation comprising a radiation sensitizing effective amount of a deoxycytidine compound selected from 5-chloro-2'-deoxycytidine and 5-chloro-2'-halo-2'-deoxycytidine, wherein halo is fluoro, chloro, bromo or iodo, together with a deamination inhibiting effective amount of a deamination inhibitor.

3. The composition of claim 2, wherein the deamination inhibitor is at least one of tetrahydrouridine or 2'-deoxytetrahydrouridine.

4. The composition of claim 2, wherein the deoxycytidine compound is 5-chloro-2'-chloro'2'-deoxycytidine.

5. The composition of claim 3, in which the weight ratio of tetrahydrouridine and 2'-deoxytetrahydrouridine to the deoxycytidine compound is from about 1:30 to 1:5.

6. A compound of the formula wherein A is NH2 or =O,X is chloro, bromo, or iodo; Y is chloro, bromo, or iodo; and ? is a single bond when A is =O, or a double bond when A is NH2.

7. A process for preparing compounds of formula I

I

wherein A is NH2 or =O; X is chloro, bromo or iodo; Y is chloro, bromo or iodo; and ? is a single bond when A=O, or a double bond when A is NH2, which comprises halogenating the 5' position of a compound of formula II

II

wherein A, Y and are as defined above.

8. The process according to claim 7 wherein the compound of formula II is prepared by halogenating the 2' position of a compound of formula III

III

wherein A and ? are as defined in claim 7.

9. A compound of formula I as defined in claim 7 whenever prepared by the process according to claim 7 or claim 8 or by an obvious chemical squivalent thereof.

10. A process for preparing compounds of formula I as defined in claim 7 wherein X represents chloro, which comprises chlorinating the 5' position of a compound of formula II.

11. Compounds of formula I as defined in claim 7 wherein X
represents chloro whenever prepared by the process according to claim 10 or an obvious chemical equivalent thereof.

12. Use of compounds of formula I as given in claim 1 for tha use of sensitizing neoplastic tissue to radiation.