CA2103050A1 - Determination of prodrugs metabolizable by the liver and therapeutic use thereof - Google Patents

Abstract

A method of ascertaining if a prodrug is useful for treating a disease is disclosed. The prodrug is acceptable if it is metabolized in liver cells by aldehyde oxidase to produce an active drug or metabolite. Prodrugs are shown equally effective in treating diseases as the active drug itself with many benefits and without as many associated side effects. Methods for treating cancers with 5-iodo-2-pyrimidinone-deoxyribose and 5-fluoro-2-pyrimidinone are also described.

Description

2 1 ~ ~ O ~i O PCI/US92/04142 DETERMINATION OF PRODRUGS METABOLIZABLE BY THE LIVER
AND THERAPEUTIC USE THEREOF

FIELD OF THE NVENTION
The present in~ention relates to prodrugs metab-olizable by the liver, and more particularly relates to treati~g disease using a prodrug metabolizable hy a li~er enzyme(s) to an active drug. It also concerns a method of ascertaining if a prodrug is useful for treati~g a disease.

BACKGROU~ QF THE INVENTION
Iododeoxyuridine (IUdR) was synthesized as an anti-neoplastic agent in 1959 by Prusoff (Prusoff, W.H., (1959), Biochem. Biophys. Acta, 32, 295-296), and was the first thymidine analog clinically used as an anti-herpes agent (Kaufman, H.E., Martola, E.L. and Dohlman, C., (1962), Archs. O~h~halmQl., 68, 235-239). The toxicities associated with IUdR when used sys~emically limited its clinical usage. IUdR was also recog~ized as a potential clinical radiosensitizer for cancer chemotherapy tginsella, T. J., Mitchell, J. B., ~usso, A., Mors~yn, G.
and Glatstein, E., (19~4~, J~ RadicLtion Qncoloqy Biol. ~
~Y~ , 1399-1406). The degree of radiosensiti~a~ion îs directly depe~de~t on the amoun~ of ~hymidine replace-ment in DNA by this analog (Speth, P. A. J., Kinsella, T.
J. Chang, A. E. Klecker, R. W., Belanger, ~. and Collins, J. M., (1988), lin. Pharmacol. Ther. 44, 369-375~.
Intrahepatic in~usion of IUdR followed by radiation for 30 the treatment of tumor cells in liver has had some success ~Remick, S. C., Benson III, A. B., Weese, J.L., Wil.lson, J. R. V.,I~Itsch, K~ D., Fischer, P~ H. and Txump, D. L., tl989), Can~er RQs. ~ 6437-6442).
In an attempt to develop selective anti-herpes 3~ simplex virus (HSV) agents based on the broader spectrum of substrate specificity of thymidine kinase of the herpes WO92/20816 PCT/US92/~142 ~ 2 simplex virus compared to the human thymidine kinase, 5-iodo-2-pyrimidinone-deoxyribose (IPdR) - which differs from IUdR by a double bonded oxygen at the 4-position of the base - was synthesized. IPdR was found to have potent activity against HSV-1 and HSV-~ in cell culture and against HSV-2 in mice (Lewandowski, G.A., Grill, S.P., Fisher, M.H., Dutschman, G.E., Efange, S.M., Bardos, T. J.
and Cheng, Y. C., (1989), Antimicrob. Aqents Chemother., 33, 340-344). This agent was not toxic to uninfected cells, nor tc mice when given orally at the dosage employed (Lewandowski, &.A., Grill, S.P., Fisher, M.H., Dutschman, G.E., Efange, S.M., Bardos, T. J. and Cheng, Y.
C., (1989), An~imicrob. A~ents Chemother., 33, 340-344) Since IPdR and IUdR are structurally related, the possible conversion of IPdR to IUdR was examined. It was ~3hown previously that lPdR could not be converted to IUdR by xanthine oxidase (Lewandowski, G.A., Grill, S.P., Fisher, M.H., Dutschman, G.E., Ef~nge, S.M., Bardos, T. J. and Cheng, Y. C., (1989), An~imic~ob. Aqents_Chemother., 33, 340-344) U.S. Patent 4,~95,937 discloses the nucleoside 1-(2-deoxy-~-D-ribofuranosyl)-5-(iodo)-2-pyrimidinone ~IPdR) for use as ~n agent against herpes viruses, for -example HSV-2. The entire content of U.S. Patent 4,~95,g37 is incorporated by reference herein.

NOMæNCLATuRE
IUdR: iodo-deoxyuridine FUdR: fluoro-deoxyuridine IPdR: 5-iodo-2-pyrimidinone-deoxyribose;
HS~: herpes simplex virus;
HPLC: high performance liquid chromatography;
IU: iodo-uracil;
EPdR: 5-ethynyl-2-pyrimidinone-deoxyribose;
IP: 5-iodo-2-pyrimidinone, BPdR: 5-bromo-2-pyrimidinone-deoxyribose;
MPdR: 5-methyl-2-pyrimidinone-deoxyribose;

W092~20816 2 l ~ 3 0 .~ O PCT/US92/~142 EtPdR: 5-ethyl-2-pyrimidinone-deox~ribose;
BUdR: 5-bromo-deoxyuridine dR: deoxyribose HBV: hepatitis B virus FU: 5-fluoro uracil FP: 5-fluoro-2-pyrimidinone ddI: dideoxyinosine ddG: dideoxyguanine DHPG: ganciclovir (9-[(1,3-dihydroxy-2-propoxy)methyl]guanine) ACV: ~S)-N-[N-(5-amino-5-carboxy-l-oxopentyl)-L-cysteinyl]-D-valine D4T: 2,3'-dideoxy-2',3'-didehydrothymicline AZT: 3'-aæido-3'-deoxythymidine $UMMARY OF THE INVENTION
It is an object of the present invention to provide compositions for use as prodrugs which are metabo-lizable in the mammalian liver into a biologically acti~e substance, particularly a biologically acti~e substance which is intended to exert its biological effect in the liver or one which cannot be administered orally.
~ It is another object of the present invention to provide compositions comprising 5-substituted PdR analogs, particul~rly PdR, for use a prodrugs which are metabo-~izable in the mammalian liver (especially the human liver) to form in situ he correspQnding biologically active 5-subs~ituted UdR compounds.
It is a further object of the present invention 3~ ~o use such 5-substituted PdR analo~s for treatment: of liver-associated diseases and particularly as a radiosen-sitizer for hepato-carcinoma.
It is still a further object of the present invention to provide compositions other than 5~substituted 3~ PdR analogs for use as ~rodrugs which are metabolizable in the mammalîan li~er into a biologically active substance, particularly a biologically active substance which is WO92/20816 PCT/US92/~142 3~3~ ~

intended to exert its biological effect in the liver or one which cannot be administered orally.
It is yet a further object of the present inven-tion to provide such compositions other than 5-substituted PdR analogs which are 5-substituted pyrimidinone analogs, particularly FP.
It is still another object of the present inven-tion to provide such compositions other than S-substituted PdR analogs which are prodrugs for the formation of bio-logically active nucleosides or nucleoside bases otherthan UdR a~d U, preferably analogs of guanosine, cytidine, inosine or thymine.
It is ano~her object of the present invention to provide for improvements in the treatment of dislease.
It is a further object of the present invention to provide a method of ascertaining if a prodrug is useful-for treating a disea~e.
It is another object of the present invention to provide a method of determining whether a prodrug is metaboliæable by a liver enzyme to a biologically active substa~ce acting on any cell in the body. .
It is another object of the pre~ent invention to provide a method of treating a di~ease using a prodrug..
It is yet another object of the present inven-tion ~o provide a method for synthesizi~g a chemical compound using an aldehy~e oxidase enzyme.
The above objects, as well as other objects, aims and advantages are satisfied by the present inventio~.
The present invention coscerns a method of ascertaining if a prodrug is useful for treating a disease in, for example, a mammal, and preferably a human, com-prising determining whether or not a non-toxic prodrug is metabolized in liver cells, in vitro, by the enzyme hepat-ic aldehyde oxidase, wherein if the prodrug is metabolized into an a~tive drug or other useful metabolite, it is an effective prodrug for use in treating a disease. The WO92/20816 PCT/US92/~142 2:~1)31~5~

determination and use of prodrugs effective against liver associated and neoplastic diseases are of particular interest.
The present invention is also directed to a method of treating a disease in an animal comprising administering to the animal, preferably a human, a pharma-ceutically effective amount of a nontoxic nucleoside analog or nucleoside base analog, or a salt or ester thereof, either alone or in admixture with a pharmaceuti-cally acceptable carriex, the analog prodrug being capableof being metabolized in liver cells by aldehyde oxidase into an active drug or other useful metabolite.

BRIEF DE~CRIPTION_ OF THE DR~WINGS
Figure 1 depicts HPLC profiles of the conversion of IPdR to IUdR by liver hor.logenate. IPdR was incubated wi~h rat li~er homogenate. For control reactions, a portion of the supernatant was boiled for 5 minutes to inactivate all enzymes before use. The assay condition was as described hereinafter except 60 ~l of the lOO,OOOg x 60 minute supernatant (equivalent to approximately 0.5 ~g protein/ml~ was~used in a reaction volume of 1500 ~l.
Aliquots (300 ~l) were removed at various time points (~
minutes, 15 mlnutes and 30 minutes from (l) to (3~ respec-tively) during the incubation period at 37C. The re~en-tion times were approxImately 9.5 minutes for IPdR(A), 8.0 minutes for IUdR(~) and 6.5 minutes for IU(C~
respec~i~ely.
Figure 2 depic~s the survival rate of mice given 100 mgjkg daily oral dosages of FU and FP for five days.
Figure 3 depicts the survival rate of mice treated with varying dosages of FU and FP given orally.
Figure 4 depicts the survival rate of mice previously injected with leukemia cells and later adminis-tered varying concentrations of FU and FP given orally.
~ igure 5 depicts the change in weight of a colontumor treated with various dosages of FU and FP given WO92/20816 PCT/US92/~142 ~30S~

orally over time. All weight figures are gi~en compared to an initial control.
Figure 6 depicts the relati~ely low IPdR incor-poration into ~issues of athymic nude mice in bone marrow and gut, wherein thymidine replacement plateaus at about 250 mg/kg/d.
Figure 7 depicts the comparative IPdR replace-ment into liver and tumor tissues of athymic nude mice between liver tissue and metabolic tumors, wherein liver incorporation was negligible compared to increasing tumor incorporation.
Figure 8 depicts results of FP treatment of female transgenic mice expressing the SV40 large tumor antigen and developing ~rarious cancers, wherein FP treated mouse lived longer and had slower increase in body weight than a control.
Figure 9 depicts results of FP treatment of male transgenic mice expressing the SV40 large tumor antigen and developing various cancers, wherein FP treated mouse had a slower increase in body weight indicating longer sur~ival than control.
.~
DETAILED DE$CRIPTION OF THE INVENTION
The prese~t invention is based on ~he discovery ~5 ~hat prodrugs are activated by liver aldehyde oxidase ln vitro and ln ~lvo and become active drugs or metabolites to achieve a high selectivity and therapeutic index. The above is depicted by che following reaction scheme:
aldehyde oxidase Prodrug ~ active drug or metabolite One reaction scheme of the above reaction scheme i9 as follows:

WO 92/20816 PCr/US92/04142 21~ 0 ~ ~

O~ Aldehyde Oxidase J~

R~

wherei~ R is I, ~, Br, Cl, ~ C~3, -ORl, -C~3, N2 ~ SRl, - C~sCR2 ~3, _ ~R2 or -N=N~ -~r, Rl is an alkyl sroup r -om to 5 ~arbon at~ms, preferably ha~ g one carbor. atom, R2 10 a~d R3, i~depende~tly of each other, are hydroge~, a C.-C. - al3cyl group or 2 haloge~ d R ' is hydroge~, a suga- residue such as ribose or deoxy~ibose, ^ C~2 - - C~2 - C:i2 OE, - C~I2 - - CE ( Cs~2 OE), SllbS ~ ~ -.uted or u~substituted al3~ ryl, cycloalkyl, cycloaryl ~; or any other desired residue which is ~ot of su~h size as to sterically hinder the actio~ of the hepatic aldehyde oxidase. It has been shown that this residue does not ~r~ter.ere with the desired actio~ of the hepatic aldehyde oxidase o~ the c c ound.
Other examDles of reaction schemes a~e as foilows:

~ ~ Aldehyde Oxidase 25 H2NJ~N N H2NJ~N N

~ ~ Aldehyde Oxidase N~

R' R' SUBSTITUTE SHEET

3~

O ~ O

5 ~ ~ 3 Aldehyde O-_dAse N

R' whe~ein R~ is as defi~ed above.
The selection of prod-ugs w~ich can be tes.ed or used in accordance with the present in~e~tion is limite~
o~ly by the structure of the acti~e d~us being produc~d by 1~ ~he action of the hepatic aldehyde oxidase. Repres~n.a-tive examples of use~ul prodrugs include ~ucleoside ana-logs and nucleoside base a~alogs. I~ th~s situatio~, the metabolized prodxug is con~erted into a product which is toxic to or is otherwise taken up so as to exer~ a desired biol~gical effect (such as radiose~sitiz2tio~) o~ly on proliferating ce~ls (sueh as cance~ cells) or to repli-cating ~irus, but is ~ot toxic to or at least is less toxic to J OX does ~ot ~xe~t the same biolosical e fect or less of the s~me ~iological effec~ on, nonprolifP-atins cells.
~ epatic aldehyde oxidase is widesprea~ amo~g m2mmalian species. This enzy~e ca~alyzes the oxidation of a va~iety of aliphati~ and aro~tic aldehydes as well as a number of non-aldehyde heterocyclic comoounds such as N~-methylnicotiuamide, 4-am~no-antifolates and me~hotrexate and its analogs. The finding by the present i~ventors OL
the oxidation of 5-substituted pyr~micinones to their uracil or uridine countex~arts by hum2n or rat alàehyde :oxidzse has resulted in a comDlete new category of substrates acted upon by this e~zyme. This discove~Y zlso allows the designi~g o~ c~ugs, met~bclized in the liver, SUBSTITUTE SHEET

WO92/20X16 PCT/US92J~142 2 1 0 3 f~ ~:i O

which can suppress and destroy cancer cells, viruses, parasites and other unwanted microbial pathogens.
To design prodrugs useful in the present inven-tion, one needs only to select a bio-affecting compound, i.e., a compound exerting a desired biolog~cal effect, which compound has a keto group in its structure and which compound is desired to be formed in situ in the liver of an animal, particularly a mammal and most particularly a human. Any such bio-affecting compound having a keto group in its structure is a potential candidate for the present invention. Once such a bio-affecting compound is selected, a simple in vltro assay may be conducted to determine whether a correspon~ing prodrug will be oxidized to the bio-affecting compound in question by hepatic aldehyde oxidase.
The prodrug is formed by synthesizing a compound corresponding to the desired bio-affecting compound but ~he keto group thereof ~eing in a reduced form. This potential prodrug is then subjected to an 1n vitro assay similar to that described in Examples 2 and 3 herein to detenmine whether it acts as a substrate for hepatic liver oxidase. If the prodrug is converted to the predetermined bio-affecting compound by the hepatic li~er oxidase, then that prodrug may be oonsidered a compound in accordance with the present inven~ion and may be used in accordance with the present in~ention.
While 5-substituted PdR analogs are preferred and have been shown to con~ert to correspondingly substi~
~uted UdR compounds in vivo, it has also been establlshed that the deoxyribose unit is not necessary foT^ substrate specificity and that FP is also metabolizable to FU b~
hepatic aldehyde oxidase in vitro as well as ln vlvo. As most nucleosides and nucleoside bases include keto groups in their formula and many are structurally related to the s~ructure of uracil, it is expected that bio-affecting compounds which are analogs of nucleosides and nucleQslde bases are prime candidates for bio-affecting compounds for WO92~20816 PCT/US92/~142 q~`~3~ - lo-which prodrugs in accordance with the present invention can be designed, metaboli~able by hepatic aldehyde oxidase into such compounds. Thus,~in addition to uracil and uridine analogs, analogs of other nucleosides and nucleo-side bases,'such as cytidine, guanosine, 6-azauridine and 8-azaguanine analogs, may be used as the basis of the formation of prodrugs in accordance with the present invention. Examples of bio-affecting compounds in this category include ddG, DHPG, ACV, ddI, D4T and AZT.
Further examples of commercially availc~le analogs of pyrimidines and purines which could serve as bio-affecting drugs for the design of correspondi.ng prodrugs according to the present invention are ]isted in Tables I and II:

TABLE I ~PYRIMIDINE ANALOGS
N'-Acetylcytidine 3'-O-Acetylthymidine ~denosine N'-Oxide Allopurinol Riboside 4-Amino-5-Aminomethyl-2-Methyl-Pyximidi~e l-Aminobarbituric Acid 2-Amino-5-Bromo-6-Methyl Pyrimidinol 4-Amino-5-Carbethoxy-2-Ethyl-Mercaptopyrimidine 5-Amino-6-Carboxy-2,4-Dihydroxy-Pyrimidine 2-Ami~o-4-Chloro-6-Methyl-Pyrimidine 3'-Amino-3'-Deo~ythymidine 5'-Amino-5'-Deoxythymidine 5'-Amino-2'-Deoxyuridine 5'-~mino-2',5'-Dideoxy-5-Iodocytidine 5'-Amino-2',5'-Dideoxy-5-Iodouridine 4-Amino-2',6-Dihydroxy-5-NitrosoPyrimidine 2-Ami~o-4,6-Dihydroxypyrimidine 4-Amino-2,6-Dihydroxypyrimidine 5-Amino-2,4-~ihydroxypyrimidine 4-Amino-l,3-Dimethyl-2,6-Dioxy-5-Nitxosopyrimidine 2-Amino-4,6-Dimethylpyrimidine 4-Amino-2-Hydroxy-5-Hydroxy-Methylpyrimidine 4-Amino-6-Hydroxy-2-Mercapto-5-Nitrosopyrimidine 4-Amino-6-Hydroxy-2-Mercapto-Pyrimidine 2-Amino-4-Hydroxy-6-Methylpyrimidine 4-~mino-2-Hydroxy-5-Methylpyrimidine 2-Amino-4-Hydroxypyrimidine 4-Amino-2-Hydroxypyrimidine 4-Amino-6-Hydroxy-2-Thiopyrimidine 2-Amino-4-Methylpyrimidine W092~20816 2 1 0 ~ O ~j O PCTJUS92/04142 4-Aminoorotic Acid 4-Amino-2-Thiopyrimidine 6-Amino-2-Thiouracil 5-Amino-2,4,6-Trihydroxypyrimidine 4-Aminouracil 5-Aminouracil 6-Aminouracil 5-Aminouricine Amobarbital 2,3'-Anhydrothymidine 5-Azacytidine 6-Azacytidine 5-Azacytosine 6-Azacytosine 5-Aza-2'-Deoxycytidine 6-Aza-2'-Deoxyuridine 6-Aza-2-Thiothymine 6-Azathymine 5-Azauracil 6-Azauracil Riboside 6-Azauridine 2'-Azido-2'-Deoxycytidine 3'-Azido-3'-Deoxythymidine 2'-Azido-2'-Deoxyuridine Barbituric Acid 3'-0-~enzoylthymidine 5'-Benxoyluridi~e 5-Bromocytidine 5-Bromocytosine 5-Bromo-2'-Deoxycytidine 5-Bromo-2,3'-Dideoxyuridine 5-~romo-2,4-Dihydroxypyrimidine ~-Bromo-2',3'-Isopropylidene-Uridine 5-Bromo-l-Methyluracil 5-Bromoorotic A~id 5-Bromouracil 5-Bromouridine ~- .
(E~-5-(2-Bromovinyl)Uridine 3-Butyluracil 5-Carbethoxycytosine 5-Carbetho~y-2,4-Dihydroxypyrimidine 5-Carbethoxy-2-Ethylmercapto-4-Hydroxpyrimidine 5-Carbethoxy-2-Thiouracil 5-Carbethoxyuracil 5-Carboxycytosine 5-Carboxy-2,4-Dihydroxypyrimidine 6-Carboxy-2,4-Dihydroxypyrimidine 5-Carboxy-2-Ethylmercapto-4-Hydroxypyrimidine 5-Carboxy-4-Hydroxy-2-Thiopyrimidine Carboxymethyluracil 6-Carboxy-5-Nitro-2,4-Dioxypyrimidine 5-Carboxy-2-Thiouracil 5-Carboxyuracil 5-Chlorocytosine Arabinoside S'-Chloro-5'-Deoxycytidine 2'-Chloro-2'-Deoxy-4-Thiouridine WO92/20816 PCT/US92~142 3~

2'-Chloro-2'-Deoxyuridine 5'-chlorodeoxyuridine 2-Chloro-4,5-Diaminopyrimidine 6-Chloro-2,4-Dimethoxypyrimidine 5 2-Chloropyrimidine 5-Chlorouracil 4,5-Diamino-2-Chloropyrimidine 4,5-Diamino-2,6-Dihydroxypyrimidine 2,5-Diamino-4,6-Dihydroxypyrimidine 10 4,6-Diamino-2-~thylmercaptopyrimidine 4,6-Diamino-5-(Formylamino)-Pyrimidine 4,5-Diamino-6-Hydroxy-2-Mercaptopyrimidine 4,6-Diamino-2-Hydroxy-5-Nitrosopyrimidine 4,5-Diamino-6-Hydroxypyrimidine - 2,4-Diamino-6-Hydroxypyrimidine 4,6-Diamino-2-Hydroxypyrimidine 4,6-Diamino-2-Methylmercaptopyrimidine 2,4-Diamino-6-Methyl-5-Nitropyrimidine 4,5-Diamino-6-Methyl-2-Thiopyrimidine 20 2,4-Diamino-5-Nitropyrimidine 4,5-Diaminopyrimidine 4,5-Diamino-2-Thiopyrimidine 4,5-Diamino-6-Thiopyrimidine 4,6-Diamino-2-Thiopyrimidine 25 5,6-DiaminouraCil 5-Diazo-2~-Deoxyuridine 5-Diazouracil 4,6-Dichloro-5-Aminopyrimidine 2,4-Dichloro-6-Methylpyrimidine 30 2,4-Dichloropyrimidine 4,6-Dichloropyrimidi~e 2',3'-Dideoxycytidine 2',3'-Dideoxyuridine 2,4-Diethoxypyrimidine 35 5,6-Dihydrodeoxyuridine 5,6-Dihydro-2,4-Dihydroxy-6-Methylpyrimidine -5,6-Dihydro-2,4-Dihydxoxypyrimidine Dihydro-6-Methyluracil Dihydrothymidine Dihydrothymine Dihydrouracil Dihydrouridine 2,6-Dihydroxy-4-Amino-5-Nitrosopyrimidine 2,6-Dihydroxy-4-Aminopyrimidine 2,4-Dihydroxy-6-Methyl-5-Nitropyrimidine 2,4-Dihydroxy-6-Methylpyrimidine 2,4-Dihydroxy-5-Nitropyrimidine 4,6-Dihydrox~-5-Nitroso-2-Thiopyrimidine 4,6-Dihydroxypyrimidine 2,4-Dihydroxypyrimidine-6-Methylsulfone 2,4-Dihydroxy-2-Thiopyrimidine 1,5-Dimethylcytosine N,N-Dimethyl-2'-Deoxycytidine 1,3-Dimethyluracil 5,6-Dioxyuracil ~.

WOg2/20816 PCT/US92/~142 21~31) .3 0 2,4-Dithiopyrimidine 3,N'-Ethenocytidine 5-Ethyl-2'-Deoxyuridine 2-Ethymercapto-4,6-Diaminopyrimidine 5 5-Fluoro-2'-Deoxyuridine Hexobarbital 5-Hydroxymethyl Cytosine 5-Hydroxymethyl-2'-Deoxyuridine 4-Hydroxy-6-Methyl-2-Thiopyrimidine 10 5-Hydroxymethyluridine 4-Hydroxypyrazolo-(3,4-d)Pyrimidine 2-Hydroxypyrimidine 4-Hydroxypyrimidine 4-Hydroxy-2-Thiopyrimidine 15 5-Hydroxyuracil S-Hydroxyuridine 6-Hydroxyuridine 5-Iodocytidine 5-Iodocytosine 20 5-Iodo-2'-Deoxycytidine 5-Iodoorotic Acid 5-Iodouracil 5-Iodouridine 2',3'-0-Isopropylidenecytidine 25 2',3'-Isopropylideneuridine 5'-Triphosphate 5-Mercaptouracil 2'-0-Methylcytidine 3'-0-Methylcytidine 5-Methylcytidine 30 5-Methylcytosine 5-Methyl-2'-Deoxycytidine ,5-Methyl-2-Thiocytosine 4-Methyl-2-Thiouracil 2-0-Methylthymidine 35 3-Methylthymidine 4-0-Methylthymidine --1-Methyluracil 3-Methyluracil 6-Methyluracil 40 2',0-Methyluridine 3-Methyluridine 3'-0-Methyluridine 5-Methyluridine 5-Nitrobarbituric Acid 45 5-Nitro-6-Methyluracil 5-Nitroorotic Acid 5~Nitrosothiobarbituric Acid 5-Nitroso-2-4-6-Triaminopyrimidine 5-Nitrouracil 50 3'-Oxauracil 5-Propyl-2-Thiouracil 6-n-Propyl-2-Thiouracil RibavirinT M
5-Sulfaminouracil S5 2-Sulfanilamidopyrimidine W092/20816 ~30~o PCT/US92/04142 Tetrahydrouridine 2-Thio-6-Azauridine 2-Thio-5-Carboxyuracil 2-Thiocytidine 2-Thiocytosine 4-Thio-2'-Deoxyuridine Thiomethyluracil 2-Thiopyrimidine 2-Thiouracil 5-Thiouracil 2-Thiouracil-5-Carboxylic Acid 4-Thiouridine 2,4,5-Triamino-6-Hydroxypyrimidine 4,5,6-Triamino-2-Hydroxypyrimidine 2~4~6-Triamino-5-Nitrosopyrimidine 2,4,6-Triaminopyrimidine 4,5,6-Triaminopyrimidine 2,4,6-Trichloropyrimidine Trifluorothymidine 2,4,5-Trihydroxypyrimidine UramilT M

TABLE I~: PURINE ANALOGS
3'-0-Acetyl-2'-Deoxyadenosine 3'-0-Acetyl-2'-Deoxycytidine N'-Acetyl-2'-Deoxycytidine N'-Acetylguanine 2-Amino-6-~enzylmercaptopurine 2-Amino-6-Benzylthipurine 2-Amino-B-Bromo-6-Hydroxypurine 2-Amino-6(~-Carboxyethyl)-Mercaptopurine 2-Amino-6-Carboxymethyl-Mercaptopurine ~-2-Amino-6-C~loropurine 2-Amino-6-Chloropurine Riboside 6-Amino-2,8-Dihydroxypurine 8-Aminoguanosine 2-Amino-6-Mercaptopurine 6-Amino-2-Methylpurine 6-Amino-3-Methylpurine 2-Aminopurine 8-Azaxanthine 8-Azidoadenosine 6-Benzylaminopurine 6-Benzylaminopurine Riboside l-Benzylinosine 6-Bromoadenine 8-Bromoadenosine 5~ 8-Bromo-2'-Deoxyguanosine ~-Bromoguanine ~-Bromoguanosine : ~-Bromoinosine 21030.i'J

6^Bromopurine 6-Carboxymethymercaptopurine 2-Chloroadenosine 5'-Chloro-5'-Deoxyadenosine 5 5'-Chloro-5'-Deoxyinosine ~-Chloro-2,6-~ihydroxypurine 6-Chloroguanine 6-Chloroguanine Riboside 6-Chloroguanosine 10 6-Chloropurine 6-Chloropurine Riboside - 8-Chloroxanthine CordycepinT ~
6-Cyanopurine 15 2,6-Dichloropurine 2'-3'-Dideoxyadenosine - 2'-3'-Dideoxyguanosine 2,8-Dihydroxyadenine 2,6-Dihydroxy-l-Methylpurine 20 2~6-Dihydroxypurine 2,6-Dihydroxypurine 6-Dimethylaminopurine 6-Dimethylam;nopurine-9-Riboside l,l-Dimethylguanidine 25 1,7-Dimethylguanine 1,7-Dimethylguanosine N~-Dimethylguanosine 1,7-Dimethylxanthine 3,7-Dimethylxanthine 30 2,8-Dithio-6-Oxypurine ~: ~,6-Dithiopurine ~l,N'-Ethenoadenosine 6-Ethoxypurine 9-Ethyladenine 35 5'-(N-ethyl)-Carboxamidoadenosine 9-Ethylguanine ~~
6-Ethylmercaptopurine 6-n-Heptylmercaptopurine 6-n-~exylamlnopl~rine 40 6-Histaminopurine N'-(2-Hydroxyethyl)Adenosine 6-(~-Hydroxyethylamino)Purine l-Hydroxy-iso-Gua~ine 2-Hydroxy-6-Mercaptopurine 45 6-Hydroxy-2-Nercaptopurine 2-Hydroxy-6-Methylpurine 6-Hydroxy-l-Methylpurine 2-Hydroxypurine 6-Hydroxypurine 50 2-Hydroxy-6-Thiopurine 6-Hydroxy-2-Thiopurine 5'-Iodo-5'-Deoxyadenosine 6-Iodopurine ~: N'-(~'-Isopentenyl)Adenosine 6-Isopropoxypurine W092/20816 PCT/US92/~142 ~o3~o 2',3'-0-Isopropylideneadenosine 2',3'-0-Isopropylideneguanosine 2',3'-0-Isopropylideneinosine 2',3'-0-Isopropylidene-6-Thioinosine 2-Mercaptoinosine 2-Mercaptopurine 6-Mercaptopurine ``
6-Mercaptopurine Arabinoside 6-Mercaptopurine 2'-Deoxyriboside 6-Mercaptopurine Riboside 2-Mercaptopyrimidine 6-Methoxypurine 6-Methoxypurine Riboside l-Methyladenine 2-Methyladenine 3-Methyladenine l-Methyladenosine 2'-0-Methyladenosine 3l-0-Methyladenosine 6-Methylaminopurine l-Methylguanine 7-Methylguanine l-Methylguanosine 2'-0-Methylguanosine 3l-0-MethylguanoSine 7-Methylguanosine l-~ethylhypoxanthine l-Methylinosine 7-Methylinosine Methylmercaptoguanine : 6-Methylmercaptopurine ~6-Methylmercaptopurine Riboside 6-Methylpurine 6-n-Propoxypurine 6-n-Propylmercaptopurine 6-Selenoguanosine --6-Selenoinosine 6-Selenopurine 6-Thioguanine 6-Thioguanosine 8-Thioguanosine Thiohydroxypurine 2-Thioxanthine 6-Thioxanthine 4~ 2,6,~-Trirhloro-7-Methylpurine 2,6,8-Trichloropurine l,3,9-Trimethylxanthine 2,6,8-Trioxypurine Examples of prodrugs which may be metabolizable by hepatic aldehyde oxidase to a corresponding uridine, WO 92~20816 P~/US92/04~4~
~1~30SIJ

thymi dine, cytidine, guaslosine, 8 - azagua~ e or 6 -a~auradine aIlalog are the following:

; 0 ~ ~ J\\~ N /

R ' H2N N\N

I~)H2 2s ~J or ~

R' R' wherein R is I , F , Br , Cl , ~ 3, - ORl, - CF3, NO2, SRl, 2 R3, - C CR2 or -N-N~ -~, Rl is an alkyl group f rom 1 to 5 carbo~ ato;ns, preferably ha~ing oD.e carbon atcm, R2 and R~ depe~de~tly o~ each oth~r, are hydrogen, a Cl-C~-alkyl group or a halogen, a~d R~ is hydrogen, a sugar residue such as ribose or deoxy-ribose, - CH2-O-C~i2-CH20~-, -CH2-O~ 0~;), substi.u.~d o-W092/20816 , PCT/US92/~142 S~

--substituted alkyl, aryl, cycloalkyl, cycloaryl or anv c_:~er desired residue which is not of such size as to c_e.ic~lly hinder the action o~ the hepatic aldehyde c~idase. It has bee~ show~ that this residue does not _~terfere with the desired action of the hepatic aldehyde cxidase on the compound.
Preferred groups for -CH=CR2R3 are -CX=CF2 a~c.
-Ci=C~2. R sugar analogs are preferably HO ~ /o~
HO~ ~ HO~ --HO~ HO~

~ ~¦ 0 2s J OH J OH ' F

30 ~ ~ ~ HO~<O-- ¦ HO~ 5~ \

Esters of the sugar analogs fcr use in the in~e~tion include esters in which X of ~OCX2 i~ the analoc is replaced by -C-~ in which the non carbonyl moiety R;
of t~e este~ group.ng is selected from hydroge~, str~is~,t SUBSTITUTE SHEET

WO92/20816 PCT/US92/~142 ~lo~o.ln or branched chain alkyl te.g., methyl, ethyl, n-propyl, t-butyl, n-butyl), alkoxyalkyl (e.g., methoxymethyl), aral-kyl (e.g., benzyl), aryloxyalkyl (e.g., phenoxymethyl), aryl (e.g., phenyl optionally substituted by halogen, Cl 4 alkyl or Cl 4 alkoxy); substituted dihydropyridinyl (e.g., N-methyldihydro pyridinyl); sulphonate esters such as alkyl or aralkylsulphonyl (e.g., methanesulphonyl);
sulphase esters; amino acid esters (e.g., L-valyl or L-isoleucyl) and mono-, di- or tri-phosphate esters.
Also included within the scope of such esters for use in the invention are esters derived from poly-functional acids containing more than one carbo~yl group, for example, dicarboxylic acids H02C(CH2)nCO2H where n is an integer of 1 to 10 (for example, succinic acid) or phosphoric acids. Methods for preparing such esters are well known in the art.
With regard to the above described esters, unless otherwise specified, any alkyl moiety present advantageously contains 1 to 16 carbon atoms, preferably 1 to 4 car~on atoms and could contain one or more double bonds. Any aryl moiety present in such esters advanta-geously comprises a phenyl group~
In particular, the ester may be a C1 6 alkyl' e~ter, an unsubstituted benzoyl ester or a ben~oyl ester substitut d by at least one halogen (bromine, chlorine, fluorine or iodine), saturated or unsaturated C1 ~ alkyl, saturated or unsaturated Cl 6 alkoxy, nitro or trifluoro-methyl groups.
Pharmaceutically acceptable salts of the above described analogs include those deri~ed from pharmaceuti-cally acceptable inorganic acids and bases. Examples of sui~able acids include hydrochloxic, hydrobromic, sulfu-ric, nitric, perchloric, fumaric, maleic, phosphoric, glycollic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic,formic, benzoic, malonic, naphthalene-2-sulfonic and benezenesulfonic acids.

WO92/20816 PCT/US92/~142 ?..~ r~3~ '?~

As used herein, the term ~analog~7 (or "active ingredient") includes the analog itself, as well as an ester or salt thereof. ~
Salts derived from appropriate bases include alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium and NR4 + (where R is Cl 4 alkyl) salts.
The choice of bio-affecting compounds for which prodrugs can be formed in accordance with the present invention is not limited solely to nucleoside or nucleo-side base analogs. Vir~ually any compound having a keto group in its structure is a candidate for a prodrug metab-olizable by hepatic aldehyde oxidase to that compound.
Such can he determined by the simple assay discussed above.
The term "bio-affecting compound" or ~'biologi-cally active substance n is intended to include compounds regulating any aspect of the metabolism of the animal to which it is to be administered or that of an organism invading the animal to which it is to be administered.
These include, without limitation, anti-depressants, antibiotics, blood-pressure regulating drugs, analgesics, anti-neoplasticsD antivirals, etc.
Therapeutic treatment of liver-associated diseases is a particularly preferred use of the prodrugs of the present in~ention due to the fact that the hepatic aldehyde oxidase was found substantially exclusively in the liver. Thus, a particular advan~age of the prodrugs of the present invention is in the elimination and reduc-tion of side-effects which would otherwise be caused by the systemic a~minlstration of the bio-affecting compound when it is desired to have its effect only in the liver.
Many compounds, such as IUdR, which are useful when treating proliferating cells, have substantial systemic toxicity which have limited their clinical utility. The corresponding prodrug generally will not have the same activity as the bio-affecting compound. Thus, such W092/20816 ~I O~ PCT/US9~/04142 prodrugs are generally non-toxic except when metabolized into the desired bio-affecting compound. For example, the 5-substituted pyrimidinone precursors for IUdR or FU are not substr~tes for human thymidine kinase and thymidine S phosphorylase and are substantially non-toxic. When used for the treatment of hepatic carcinoma, for example, IPdR
will be metabolized in the liYer by the hepatic aldehyde oxidase to IUdR which will then be preferentially taken up by the tumor cells in the liver before substantial spread ~0 of the IUdR to other tissues can occur. Thus, the therapeutic index of the 5-substituted PdR compounds for primary liver cancer or metastatic liver cancer will be much better than their UdR counterparts.
There are other occasions besides the treatment of liver-associated diseases when the administration of a prodrug will be preferred to the administration of the bio-affecting compound which i5 formed from such prodrugs.
For example, many drugs are not orally administrable for any of a variety of reasons. Oral administration of a prodrug from which the active compound is released in the li~er will avoid most of the disadvantages of oral admin-istration of the acti~e compound. Thusl for example, ~U
is a well-known anti-cancer drug which cannot be adminis-tered orally. FP is a prodrug which is metabQlizable by hep tic aldehyde oxidase to FU. FP may be given orally to a ~ubject. As is shown in the examples below, FP has demonstrated therapeutic effecti~eness n leukemia and colon cancer. The effecti~eness of FP is essen~ially the same as FU, indicating effective delivery of FU from the hepatocytes to the tumor.
Currently, IUdR and BUdR are being explored as radiosensitizers. However, their effective use is limited by their cytotoxicity and rapid catabolism to the free base, followed by dehalogenation (Speth, P. A. J., Kinsella, T. J., Chang, A. E., Klecker, R. W., Belanger, K. and Collins, J. M., (1988), Clin. Pharmacol. Ther. 44, ? S ~3 PCT/US92/~142 ~. ~

369-375). Since IPdR and its analogs are virtually non-toxic, and are not substrates for thymidine phosphorylase, the use of IPdR and its analogs instead of IUdR or ~UdR
will circumvent the difficulties of toxicity and degrada-tion related to radiation therapy.
"Liver-associated diseases" include ~iral hepa-titis, for example, hepatitis A, hepatitis B, hepatitis C
and hepatitis D; hepatoma; cancers metastasized to and from the liver; infection with cytomegalovirus or other viruses, parasitic infections, e.g. Schistosomiasis, Clonorchiasis, Fascioliasis, Opisthorchiasis; ancl infec-tion by an assortment of flukes and tapeworms, microbial agents, e.g. fungal or bacterial infections, such as Para~occidioides brasiliensis, and other liver diseases, such as cirrhosis of the liver, or rejection of liver transplants.
Metabolic conditions or diseases are also treat-able by selecti~e use of a prodrug which is metabolizable into an active compound by the hepatic aldehyde oxidase.
Drugs may be those which are targeted to receptors in or on liver cells as well as non-liver cells are also of value. These drugs may have either whole or partial agonist or antagonist activity. A great variety of receptor acting drugs may be created by administering the corresponding prodrug to a subject or to a preparation of the aldehyde oxidase in ~itro. Thus prodrugs of drugs acting at remote sites such as the heart or brain, may also be used in accordance with the present invention.
Furthermore, if liver cells lack the target receptors, the potential for unwanted toxicity due to high liver concen-trations are reduced. The use of prodrugs metabolizable by a liver enzyme to active drugs has wide applicability.
Depending on the level of enzyme activity of the aldehyde oxidase on a prodrug, a slow rate of formation or slow release of active drug into the body may be achieved.
While the compounds exemplified herein are rapidly metabo-lized, they can be modified to slow their metabolism.

WO92/20816 2 ~ 3 0 .~ O PCT/US92/04142 This should result in a requirement for less freguent dosages of prodrug compared to the drug, with all of the known advantages of patient compliance and constancy of dosage. If the metabolism to the active drug is suffi-ciently slow, entirely new classes of compounds may beused therapeutically which could not be used befoxe due to toxici~y problems with bolus dosages. It can be seen, for example, from Table V hereinbelow that the nature of the R
group on the pyrimidinone substrate substantially effects the rate of conversion. Thus, it is clear that prodrugs can be designed with a predetermined rate of metaLbolism.
One example of a drug which would be improved by slow conversion in vlvo is the ketone containing drug suramin. The effective anti-cancer dosage for metastatic prostate cancer is very close to the dosage resulting in paralysis, as was accidently and unfortunately discovered during human trials. Bolus dosages were found to be significantly less effective. As a consequence, pa~ients requixing such treatment are hospitalized and continuously monitored and infused to maintain a narrow range of effec-tive and acceptable concentrations. ~y using an aldehyde or other prodrug form of suramin metabolizable by aldehyde oxidase to active suramin, it should be possible to give the patient bolus doses without a need ror constant hospi~alization.
As indicated above, orally acceptable prodruss may be used in place of their ac~ive product which are not orally acceptable. A prodrug may differ from its active product by increased stability in acidic conditions, resistance to digestive enzymes, better adsorption or less irritation or toxicity to the digestive system. Once adsorbed, the prodrug is converted to the active drug by liver aldehyde oxidase, thereby bypassing the diges~ive track. The advantages of oral versus parenteral adminis-tration are readily apparent to those skilled in the art.
Regardless of the route of administration, anydrug has half-life limitations due to renal clearance, WO92/2081~ PCT/USg2/04~42 c~ 3~

enzymatic degradation, and too much or too little binding to serum proteins and the like. The use of prodrugs broadens the opportunities for drug development by compen-sating for such problems.
The amount of the analog described above for use in the present invention will ~ary not only with the particular compound selected, but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately determined by the discretion of the at:~endant physician or veterinarian. In general, however, a suit-able dose will be in the range from about 1 to about 100 mg/kg of body weight per day, preferably about 2 to about 50 mg per kilogram body weight per day, most preferably 2 to 10 mg/kg/day.
The desired dose may conveniently be presented in a single dose or as di~ided doses administered at appropriate intervals, for example, at two, three, four or more sub doses per day.
~0 The analog as described above is conveniently administered in unit dosage; for example, containing ~.5 to 50 mg, preferably 20 to 1000 mg, most preferably S0 to 700 mg, of active ingredient per unit dosage form.
Ideally, the active ingredient shoul~ be admin-istered to achleve peak plasma concentrations of theactive ingredient of from about 1 to 7S ~M, preferably about 2 to 50 ~M, most preferably about 3 to about 30 ~M.
This may be achieved, for example, by the intravenous injection of 0.1 to 5~ solution of the prodrug, optionally in saline, or administered as a bolus contairli~g about O . ï
to 50 mg/kg of the active i~gredient.
It will be appreciated that different prodrugs may require ~astly different dosages. Furthermore, treat-ment of diseases of different tissues or organs may also re~uire different dosages. These dosages are readily determinable by one of ordina~ skill in the art using methods known in the art.

~4. ~3~0 While it is possible that, for use in therapy, the analog described above may be administered as the raw chemical, it is preferable to present the prodrug in conjunction with a pharmaceutically acceptable carrier as a pharmaceutical formulation.
The invention thus further provides a pharmaceu-tical formulation comprising an analog as described together with one or more pharmaceutically acceptable carriers therefor and, optionally, other therapeutic and/or prophylactic ingredients. The carrier(s) must be "acceptable" in the sense of being compatible wit:h the other ingredients of the formulation and not deleterious to the recipient therefor.
Pharmaceutical formulations include those suit-able for oral, rectal, ~asal, topical (including buccal,sub-lingual and transdermal), Yaginal or parenteral (including intramuscular, sub-cutaneous and intravenous) administration or in a form suitable for administration by inhalation or insufflation. The formulations may, where appropriate, be conveniently presented in discrete dosage u~its and may be prepared by any of the methodæ well known in the art of pharmacy. All methods include the step of bringing into a~sociation the active ingredient with liguid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired fonnulation. Encapsulation of the ch~mical, such as by a liposome or vesicle, may also be used where indi-cated for delivery or stabilization purposes.
Pharmaceutical formulations suitable for oral administration may convenientiy be presented as discret~
units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution; as a suspension; or as an emulsion The active ingredient may also be presented as a bolus, electuary or paste. Tablets and capsules for oral administration may contain conventional excipients WO92/20816 ~ ~ PCT/US92/~142 such as binding agents, fillers, lubricants, disinte-grants, or wetting agents. The tab]ets may be coated according to methods well known in the art. Oral liquid preparations may be in the form of, for example, aqueous or oily suspension, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for consti-tution with water or other suitable vehicle before use.
Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils) or preservatives.
The active ingredient may also be formulated for parental administration (e.gO, by injection, for ~example, bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose contain-ers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulary agents such as suspending, stabilizing and/or dispersing agents.
Alternatively, the active ingredient may be in powder fonm, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a ' suitable vehicle, e.g., sterile, pyro~en-free water, before use.
Pharmaceutical formulations suitable for rectal administration, wherein the carrier is a solid, are most preferably represented as unit dose suppositories. Suit-able carriers include cocoa butter and other materials commonly used in the art, and the suppositories may be con~eniently formed by admixture of the active compound with the softened or melted carrier (9) followed by chilling and shaping in molds.
Formulations suitable for vagi~al administration m~y be presented as pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the ~I.a3v.'ilJ

active ingredient, such carriers as are known in the art to be appropriate.
For intra-nasal administration, the active ingredient may be used as a liquid spray or dispersible powder or in the form of drops.
Drops may be formulated with an aqueous or non-aqueous base comprising one or more dispersing agen~s, solubilizing agents or suspending agents. Liquid sprays are conveniently delivered from pressurized packs.
For administration by inhalation, the active i~gredient is conveniently delivered from an insufflator, nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray. Pressurized packs may comprise a suitable propellant such as dichlorodifluoro-methane, ~richlorofluoromethane~ dichlorotetrafluoro-ethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be deter-mined by providing a valve to deliver a metered amount.
Alternatively, for administration by inhalation or insufflation, the active ingredient may take the form of a dry powder composition, for example,'a powder mix of the compound and a suitable powder base such as lactose or starch. The powder composition may be presented in u~it dosage form iIl, for exam~le, capsules or cartridges or, e.g., gelatin or blister packs from which the powder may be admi~istered with the aid of an inhalator or insufflator.
When desired, the above described formulations adapted to give sustained release of the active ingredient may be employed.
The pharmaceutical compositions for use accord-ing to the invention may also contain other active ingre-dients such as antimicrobial agents or preser~atives.
The pharmaceutical composition for use according to the present invention may also contain inert ingredi-ents such as desiccants, substances to provide ease of WO92/20~16 ~ ~ PC~/US92/04142 3~

handling, colorants, flavorants and coatinys for easy swallowing.
The active ingredient may also be used in combi-nation with other therapeutic agents, for example, other anti-infective agents. In particular, the compounds of formula (I) may be employed together with well known anti~iral agents, e.g., adenine arabinoside or interferon a? .
The invention thus provides, in a further aspect, a method comprising the use of an analog described above with another therapeutically active agent, in par-ticular, an anti-HBV agent.
When the active ingredient generated from the prodrug is an anti-cancer agent, other anti-cancer or immunomodulating agents may be employed together, as may any other compatible combination of drugs whether the activities are synergistic, complementary of separate.
The combinations referred to above may conve-niently be presented for use in the form of a pharmaceuti-cal formulation and thus the use ~f pharmaceutical~ormulations comprising a combination as d~fined above together with a pharmaceutically acceptable carrier there-for comprise a further aspect of the invention.
The individual components of such combinations may be administered either sequen~ially or simultaneously in separate or combined pharmaceutical formulations.
When an analog as described above is used in combination with a second therapeutic agent for the same disease, e.g. acti~e against the same virus, the dose of each compound may be either the same or different from that when the analog is used alone. The appropriate dose will be readily appreciated by those skilled in the art~
The aldehyde oxidase is advantageously purified from liver and may be immobilized on a solid phase for easy separation of enzyme catalyst from a reaction mix-ture. The desired bio-affecting optically active com-pounds are then separated from procompound precursors and WO92/20816 ~1 ~ 3 ~ ~ ~ PCT/US92/04142 recovered. Techniques for enzyme purification are well known in the art, any suitable one of which may be employed. Techniques for enzyme immobilization to a solid phase whether it be adsorbed, entrapped, chemically bound or retained behind a semipermeable membrane are also well known in the art.
The contents of all references mentioned in this application are incorporated by reference. The invention will now be described with reference to the following non-limiting examples.

EX~MPLES

-Example 1: Tissue Prepara~ion Rat hepatic tissue was washed with ice-cold 1.15~ KCl and blotted dry. The tissue was then homoge-nized with a tissue homogenizer, in a volume of 1.15~ KCl that was 3 times the tissue weight, to form a 25~ (w/v) homogenate. The homogenate was then centrifuged at lO,OOOg for 10 minutes at 4C. The resulting supernatant was filtered throu~h Miracloth (similar to cheese cloth) then dialyzed overnight against 50 mM Tris-HCl buffer pH
7.5 and stored at -80C before use. Rat hepatocytes w~re obtained by a perfusion technique then the cells were extracted with 10 mM phospha~e buffer, pH 7.5, containing 1 M KCl and dialyzed for 4 hours against 50 mM Tris-HCl buffer pH 7.5.

Example 2- Assay conditions For the standard assay condition, the reaction mixture contained 50 mM Tris-HCl, pH 7.5, 1 I~M EDTA, 180 ~M of IPdR (or its analogs) and approximately 0.01 mg protein of lO,OOOg supernatant of tissue homogenate in a fi~al ~olume of 500 ~1 and the incubation was at 37C for 35 10 minutes unless specified otherwise. 300 ~l of reaction mixture was removed at the end of incubation and mixed with 630 ~l acetonitrile then agitated. The precipitated WO92/20816 ~ ~ P~T/US92/04142 ?~3~ ~`

protein were removed by centrifugation and the supernatant was lyophilized to dryness. The samples were reconsti-tuted to the original aliquot volume with the HPLC mobile phase buffer and analyzed on a Alltech RP-18 column. IPdR, IUdR and IU were detected at a W absorption wavelength of 230 nm, and IPdR was also detected at a W absorption wavelength of 335 nm. The mobile phase was 10% aceto-nitrile / 90~ mM ammonium acetate, pH 6.8 and the flow rate was 1 ml/minute. Standard curves of IPdR and IUdR
were established from the integration ~alue of known concentrations.

Example 3: Conversion of 5-iodo-2-pyrimidinone-2'_ deoxyribose (IPdR) to 5-iodo-~deoxyuridine ~IUdR) To study the conversion of IPdR to IUdR by liver enzyme the metabolites of IPdR were analyzed after incuba-tion with a supernatant of rat liver homogenate using a reverse phase HPLC technique. IPdR and IUdR could be detected at an absorption wavelength of 230 nm but only IPdR could be detected at 335 nm. In order to limit the phosphorolytic cleavage of IUdR to iodouracil (IU) by thymidine phosphorylase ~Kinsella, T. J., Mitchell, J.B., Russo, A., Morstyn, G. and Glatstein, E.. ~l984), J.
Radiation Oncology ~iol. Phys., lO, l399-l406) Tris-HCl buffer was employed in the assay. As shown in Fig. l, there was a time dependent conversion of IPdR to IUdR and IUdR appeared to be the only product produced by IPdR.
The identification of IUdR was confirmed hased on the retention time on a C-18 column (8 minutes vs. 9.5 minutes for IPdR) and the W spectru~ as well as nuclear magnetic resonance spectroscopy (results are not shown).

Example 4: Properties of "IPR oxidase" activit~
This "IPdR oxidase" activity does not rec~ire exogenous cofactors, is much less active in extracts of kidney and spleen than liver (Table III) and cannot be WOg2/208~6 PCT/US92/04142 2 I O ~ O ~ V

detected in lung or intestine extracts from rats. The human liver contains a similar amount of this enzyme.
Extracts of hepatocytes, which represents about 80~ of the cell population in liver, had a very similar specific activity compared with the whole iiver extract, suggesting that the IPdR oxidatioIl is mainly present in this cell population. Differential fractionation centrifugation was used in an attempt to localize this IPdR conversion enzyme activity in the liver homogenate. The enzyme activity assay was performed on each fraction. The only :Eraction which showed the enzyme activity appeared in ~he lOO,OOOg x 60 minutes supernatant; this suggested that th:is enzyme is only located in the soluble fraction of cytosol.
Further purification was achieved by use of DEAE cellulose column chromatography, blue Sepharose column chromatogra-phy and glycerol gradient centrifugation in this order.
This IPdR oxidase acti~ity was purified 380 fold starting from the crude extracts of rat li~er. The partially purified enzyme catalyzed IUdR synthesis a~ a rate of 3.8 ~moles per minute per milligram of protein at 37C under these conditions. The apparent molecularrweight of this -enzyme in ~oth rats and humans as determined by centrifu-gation on a 20 to 40~ glycerol gradient is approximately 280,000 dalton. Neither cofactor nor di~alent cations 2S requirement has been found ror the rat liver enzyme or the h~man liver enzyme.

WO92/~0816 ~ PCT/US92/~142 Table III. Tissue specificity of the conversion of IPdR
to IUdR by cell extracts S

Tissue ~or cells) Specific activity~
(n mole/mg protein/min) ~ ~ ~ . , .
liver (human) 2-7 liver (rat) 5-15 hepatocyte (rat) 5-ll kidney (rat) 0.8-l.6 l5 spleen (rat) 0.2-0.5 intestine (rat) c0.02b lung (rat) c0.02b .
a The specific activities were obtained from 3 samples for each tissue (also 3 samples for rat hepatocyte) except the data for rat liver which were obtained from 5 samples.
b The conversion of IPdR to IUdR by rat, lung and intestine was not detected.

Exam~le ~: Identification of "IPdR oxida~e"
In order to determine which enzyme is responsi-ble for the catalysts of this IPdR oxidation, a series of known oxido-reductases which have l3imilar capability to~
oxidize a carbon atom with an adjacent amino grQup into a carbonyl functionality were explored for their ability to catalyze IPdR oxidation. Xanthine oxidase ~Boehringer Mannheim) isolated from cow milk which catalyzes the oxidation of hypoxanthine to xanthine failed to convert IPdR to IUdR. A mixed function oxidase system prepared from the microsome of rat liver a broad spectrum of sub-strates and is NADPH dependent. However, this microsomalfraction from rat liver showed no IPdR oxidase activity no matter whether NADPH was added or not. Alcohol dehydro-genase ~which can be obtained from Sigma) and alcohol oxidase (which can be obtained from Sigma) both reside in the soluble fraction of livex cell extracts, but the conversion of IPdR to UdR was not detected with purified WO92/2081~ 3 )V PCT/US92/~142 preparation of either enzyme under the same conditions that they converted their natural substrates effectively.
Sarcosine oxidase (Boehringer ~annheim) which catalyzes the conversion of Nl-methylglycine to glycine had no IPdR
oxidase activity. Phenylalanine hydroxylase, urocanase, cy~tathionine ~-lyase, L-glutamate dehydrogenase, cysta-thionase and several other oxido-reductases were ruled out based on substrate competition assays, their different cofactor specificities or some other characteristic features from literature (Weidig, C. F., Halvorson, H. R.
and Shore, J. D., (1977), Biochemistry 16, 2916-:2921;
Kato, N., Omori, Y., Tani, Y. and Ogata, K., (19'76), Eur.
J. Biochem., 64, 341-350; Keul, V., Kaeppeli, F., Ghosh, C., Krebs, T.,Robinson, J. A. and Retey, J., (1979), J.
Biol. Chem., 254, 843-~51; George, D. J. and Phi:llips, A.
T (1970) J Biol Chem., 245, 528-537; ~rodie, B. B., ., , Axelrod, J., Cooper, J. R., Gaudette, l., LaDu, B. N., Mitoma, C. and Udenfriend, S., (1955), Science, 121, 603-604; Eeme, D., Durieu-Trautmann, O. and Cha~agner, F., (1971), Eur. J. Biochem., 20, 269-275).
Since hepatic aldehyde oxidase ~as the same molecular weight, is in the cytoso~ic fraction of li~er cells and has broad substrate specificity (Rajagopalan' K.
V., Fridovich, I. and Handler, P.l (1962), J. Biol. Chem., 237, 922-~2~, it is considered that i~ is the ~IPdR
oxidase" enzyme responsible for the conversion of IPdR to IUdR. H patic aldehyde oxidase which catalyzes the oxida-tion of a variety of aldehydes to the corresponding acids also converts Nl-methylnicotinamide (Sigma) to Nl-methyl-2-pyridone-5-carboxamide and N1-methyl-4-pyridone-3-carboxamide tRajagopalan~ K. V., Fridovich, I. and ~andler, P. (1962), J. Biol. Chem., 237, 922-928;
Rajagopalan, K. V. and Handler, P., (1964), J. Biol. Chem.
239, 2022-2035; Stanulovic, M. and Chaykin, S., (1971), Archs. of_Biochem. and Biophy., 145, 27-34; Stanulovic, M.
and Chaykin, S., (1971), Archs. of Biochem. and ~io~hy., 145, 35-42; Felsted, R. L., Chu, A. E. and Chaykin, S., ~ - 34 -(1973), J. Biol. Chem., 248, 2580-2587; Barber, M. J., Coughlan, M. P., Rajogopalan, K. V. and Siegel, L. M., (1982), Biochemistry, 21, 3561-3568; Badwey, J. A., Robinson, J. M., Karnovsky, M. J. and Karnovsky, M. L., (1981), J. Biol. Chem., 256, 3479-3486). This aldehyde oxidase activity was reported to be stimulated by potassium ferricyanide and Tris buffer but not by MgCl2 (Felsted, R. L., Chu, A. E. and Chaykin, S., (1973), J.
Biol. Chem., 248, 2580-2587. This enzyme could be inhibited by 2-mercaptoethanol, dithiothreitol and other thiol agents (Rajagopalan, K. V. and Handler, P., (1964), J. Biol. Chem. 239, 2022-2035). There was no sig~nificant inhibition by cysteine at 5 mM, however at 50 mM a potent inhibition of the enzyme activity was obse~ved (Felsted, R. L., Chu, A. E. and Chaykin, S., (1973), J. Biol. Chem., 248, 2580-2587). Divalent metal cations such as Cu++, Zn++ and Fe++ caused strong inhibition (Rajagcpalan, K.
V., Fridovich, I. and Handler, P., (1962), J. Biol. Chem., 237, 922-928). The activity could also be inhibited by acetaldehyde, but not by allopurinol or formaldehyde (Rajagopalan, K. V. and Handler, P., (1964`3, J. Biol.
Chem., 239, 2022-2035; Stanulovic, M. and Chaykin, S., (1971), Archs. of Bioçhem. and Biophy., 145, 35-42;
Badwey, J. A., Robinson, J. M., Karno~sky, M. J. and Karnovsky, M. L., (1981), J. Biol. Chem., 256, 3479-3486).
Therefore a series of compounds were examined for their effects on the oxidation of IPdR to IUdR and i~ was found that the inhibition profile of compounds with the "IPdR
oxidase" activity (Table IV) was essentially identical to ~ the characteristic pattern of aldehyde oxidase. Further-more, throughout each step of the purification, aldehyde oxidase could not be separated from "IPdR oxidase~
activity.

WO92/20816 2 1 0 3 ~ ~ o PCT/USg2/04142 Table IV. Effect of inhibitors on the oxidation of IPdR
to IUdR by liver homogenate Compound tested IC50(mM)a lO Mercaptoethanol l.8 Dithiothreitol O.l Cysteineb ~50 l-~utane thiolC ~50 S~F-525A (Calbiochem) 0.03 l5 Hydroxyl amine Hydrogen peroxide lO
Cu + 0.4 Zn++ 0.3 Fe+' 20 Acetaldehyde 5 Nl-methylnicotinamide 25 a The concentrations of each compound at 50~ inhibition of the con~ersion of IPdR to IUdR.
b Approximately 40~ product inhibition was observed at 50 mM.
c Approximately 25~ product inhibition was observed at 50 - ~.

Example 6: Substr~te s~ecificity --Several 2-pyrimidinone deoxyribose anaiogs were examined for the con~ersion to their deoxyuridine counter-parts. The Michaelis constant Km for IPdR in the-reaction at pH 7.5 and pH 9.5 is 150 ~M and 87 ~M respectively, and the Km for 5-ethynyl-2-pyrimidinone deoxyribose ~EPdR) in the reaction at pH 7.5 and pH 9.5 is 77 ~M and ~6 ~lM
respectively. Nevertheless the relative Vmax for IPdR in the reaction at pH 7.5 and pH 9.5 is the same. 5-iodo-2-pyrimidinone (IP), the aglycose of IPdR was an excellent substrate for aldehyde oxidase. The synthetic substrates WO92/20816 PCT/USg2/~142 3~ ~

for aldehyde oxidase appear to be better than its natural substrates, Nl-methylnicotinamide and acetaldehyde, as judged by the potency of inhibition of Nl-methylnicotin-amide and acetaldehyde to the IPdR oxidation reaction (Table IV). The rate of reactivity of the liver en~yme with different IPdR analogs follows the order EPdR, IP, IPdR, 5-bromo-2-pyrimidinone deoxyribose (~PdR) and 5-methyl-2-pyrimidinone deoxyribose (MPdR) or 5-ethyl-2-pyrimidinone deoxyribose (EtPdR) (Table V). Electronega-tive substituents in the 5-position seemed to increase the substrate activity in this oxidation reaction.

Table ~. Substrate specificity of the conversion of RPdR to RUdRa ~ ~ Aldehyde Oxidase H

~ R' .
~ SubstrateRate of conversionb Product R R' abbre~iation(n mole/mg/min) ~bbreviat~on C~CH dRC EPdR 20 ~4 EUdR
I dR IPdR ll ~2 IUdR
30 I H IP 18 ~3 IU
Br dR BPdR 7 i2 BUdR
CH3 dR MPdR cO,ld MUdR
CH2CH3 dR EtPdR cO.ld EtUdR

a Amount of substrate used was 180 ~M in all cases.
4 0 b Determined ba9ed on 2 to 4 separate experiments.
c C = deoxyribose d Products of the reaction from MPdR and EtPdR were not detected.

WO92/20816 PCT/US92/Wl42 2~ 030 ~0 Example ?: Toxicity of FU_and FP.
~ DF1 mice were ~dministered daily oral dosages of either 50, 75 or 100 mg/kg of FU or 100, 150 or 200 mg/kg of FP. The survival rates of each are presented in chart and graphic forms in Table VI and figures 2 and 3.
The toxicity of FP was considerably less, even when higher dosages were administered.

Table VI: TOXICITY OF FU AND FP IN ~DF1 MICE

Compound Schedule of Dose Route of Death/ LD50 Injection ~mg/kg) injection Total (mg/kg) Daily FU Day 1,2,3,4,550 p.o. 5/5 2/~ 70 FP 100 p.o. 5/5 150 4/5 ~80 ~00 Of5 , Example 8: Effec~s of FU and FP on Leukemia Cells.
Mice were injected with 100~000 ~388-R leukemia cells to induce a leukemia. These cells were resistant to Adriamycin~ Daily treatments of 25 and 50 mg/kg FU and 50 and 100 mg~kg FP were given orally to these leukemic mice and the duration of survival was measured. The results are given in tabular and graphic fo.rm in Table VII and Figure 4. The survival time was as long using FP compared to FU.

WO g2~20816 PCI`/US9~ 42 Table VII. EFFECT OF FU AND FP ON SURVIVAL TI~E OF MICE
BEARING LEUKEMIA CELLS

Group Number Treatmenta Dose ILSb Curesf of Animals (Day) (mg/kg) (~) Total _ _ _ , _ EU 5 Daily on Day 25 5 0/5 1,2,3,4,5 50 25 0/5 , Inoculum: 106 P388-R cells into each mouse (i.p.).

Treatment days are those days at which the animals received inj ections of the drug (day of tumor inoculation is Day O~
b I~S, Increase in life span over controls which is expressed in terms of dying mice.
Ex ~ le 9: Effects of FU and FP on Colon Carcinoma.
Mice were in~ected with colon 3B cells and were eithex not treated or treated with daily treatments of 2S
and 50 mg/kg FU and S0 and 100 mg/kg FP were given orally to the mice and the change in tumor weight was measured~
over time. The data is di~played in figure 5. The reduc-tion in tumor ~ize or reduction in its growth rate is comparable ~etween FU and ~P.

Ath~mic Nude Mice _ . .
Mice were administered daily oral dosages of 0, 100, 250 and 500 mg/kg. Percent dThd replacement as indicative of IPdR incorporatioIl into boIle marrow, gut and liver tissue was assayed according to known methods. As pre~ented in Figure 6, relatively small amounts of thymid-ine was replaced by IPdR in bone marrow and gut, with thymidine replac~ment plateaus occurring at treatment levels above 250 mg/kg/d. No incorporation of IUdR into liver was found. Results presented in Figure 6 are mean WO92/20816 ~ O~ ~ PCT/U~92/04142 percent thymidine replacement ~ one standard error of the mean. N is greater or equal to 3 for each dose. As shown by these results, no appreciable thymidine replacement by IUdR occurred in liver, and very small percent replacement was found in bone marrow and gut. Accordingly, these results establish that the administration of IPdR used as a prodrug according to the present invention should be suitable for treatment of mammals including humans.
y Example ll: IPdR Incorporation into Tissues of ~hYmis Nude Mice Having Me~astatic Tumors _ Mice were admini~tered daily dosages oi, 0, lO0, 250 and 500 mg/kg/d to determine thymidine replacement in liver and tumor tissue. The results presented in Figure 7 are presented as mean percent thymidine replacement ~ one standard erxor of the mean. n 2 3 (control tumors n=~).
While a very small percent incorporation was detected in ~ormal liver, an increasing percent replacement was shown in the tumor, thu~ demonstrating that the use of IPdR as a prodrug for tumor treatment is expected to have good results.

Example 12: Treatment of Transgenic Mice by FP
Transgenic mice obtainecl according to the method of Sepulveda et al., Cancer ~esearch 49:6108-bll7 (1989) were administered beginning at nine weeks after birth in a female and male, whexein the treatment group received lO0 mg/kg two times per day, once per week. Figures 8 and 9 show the change in body weight over weeks 9-~7 for female and male mice, respectively. Both male and female treated mice showed a signifi_antly decreased weight gain after week 13, which correlates to increased survival time.
The female treated rat survived the control rat which died of cancer complications at week 16. Accordingly, the abo~e data suggests that the use of FP as a tumor treat-ment according to the presently claimed invention is expected to provide good results.

W092/2~16 ~3~ ~ PCT/US92/04142 The references cited above are all incorporated by reference herein, whether specifically incorporated or no~. U.S. priority applic~tions 07/701,462, filed May 15, 1991, and 07/829,474, filed February 3, 1992, are also both incorporated herein by reference.
Having now fully described this in~ention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equi~alent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.
While this invention has been described, in connection with specific embodiments thereof, it will be understood that it is capable of further modifications.
This application is intended to cover any ~ariations, uses, or adaptations of the inventions following, in general, the principles of the invention a~d including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows'in the scope of the appended claims.

Claims (15)

WHAT IS CLAIMED IS:

1. In a method of obtaining a desired biological effect in an animal by delivering an effective amount of a bio-affecting compound to the desired site in the animal, said compound having a keto-group in its structure, the improvement wherein said delivering step comprises administering to the animal a prodrug having a structure corresponding to that of said compound except that the keto group is reduced, said prodrug being capable of being oxidized to said compound by hepatic aldehyde oxidase, wherein said compound is formed in situ in the liver of the animal by oxidation of said prodrug by means of hepatic aldehyde oxidase.

2. A method in accordance with claim 1, wherein.
said bio-affecting compound is a 5-substituted UdR
compound and said prodrug is a corresponding 5-substituted PdR compound.

3. A method in accordance with claim 2, wherein said bio-affecting compound is IUdR and said prodrug is IPdR.

4. A method in accordance with claim 1, wherein said bio-affecting compound is other than a 5-substituted UdR compound.

5. A method in accordance with claim 1, wherein said bio-affecting compound other than a 5-substituted UdR
compound is an analog of a nucleoside or nucleoside base.

6. A method in accordance with claim 5, wherein said bio-affecting compound is a 5-substituted uracil.

7. A method in accordance with claim 1, wherein.
said prodrug is selected from the group consisting of:

and wherein R is I, F, Br, Cl, H, -CH3, -OR1, -CF3, NO2, SR1, -CH=CR2R3, -C?CR2 or -N=N+ -N-, R1 is an alkyl group from 1 to 5 carbon atoms, preferably having one carbon atom, R2 and R3, independently of each other, are hydrogen, a C1-C5-alkyl group or a halogen, and R1 is hydrogen, a sugar residue such as ribose or deoxy-ribose, -CH2-O-CH2-CH2OH, -CH2-O-CH(CH2OH), substituted or unsubstituted alkyl, aryl, cycloalkyl, cycloaryl or any other desired residue which is not of such size as to sterically hinder the action of the hepatic aldehyde oxidase.

8. A method in accordance with claim 7, wherein in said prodrug, R1 is , , , , , , , , , , , , or

9. A method in accordance with claim 2, wherein said biological effect is the treatment of hepato-carcinoma.

10. A method in accordance with claim 1, wherein said biological effect is the treatment of a liver-associated disease and said bio-affecting compound is one effective for the treatment of said liver-associated disease.

11. A method in accordance with claim 10, wherein said animal is a human.

12. A method in accordance with claim 1, wherein said bio-affecting compound is one which cannot be effectively administered orally and wherein said prodrug is administered orally.

13. Use of a prodrug having a structure corresponding to that of a bio-affecting compound having a keto group in its structure, except that in said prodrug the keto group is reduced, said prodrug being capable of being oxidized to said compound by hepatic aldehyde oxidase, for the manufacture of a medicament for obtaining the desired biological effect obtained by administering said bio-affecting compound.

14. A prodrug having a structure corresponding to that of a bio-affecting compound having a keto group in its structure, except that in said prodrug the keto group is reduced, said prodrug being capable of being oxidized to said compound by hepatic aldehyde oxidase, when used for obtaining the desired biological effect obtained by administering said bio-affecting compound.

15. A method of ascertaining if a substantially non-toxic prodrug is effective for treating a disease, comprising determining whether or not said prodrug is metabolized in vitro by hepatic aldehyde oxidase to a bio-affecting compound by contacting the prodrug with the hepatic aldehyde oxidase and measuring whether or not the prodrug is metabolized into a bio-affecting compound, wherein if the prodrug is metabolized into a bio-affecting compound, it is an effective prodrug for treating a disease.