CA2239203A1 - Drug therapy - Google Patents

Abstract

A therapeutic system comprising: (a) a compound comprising a target cell-specific portion and a portion capable of converting a substance into another substance; and (b) a molecule capable of substantially inhibiting the conversion of said substance, or a precursor of said molecule. In one particularly preferred embodiment said other substance is cytotoxic and said substance is substantially non-cytotoxic, the system further comprising said substance. In a second particularly preferred embodiment said substance, in its native state, is able to inhibit the effect of a cytotoxic agent and said other substance has less effect against said cytotoxic agent, the system further comprising (a) a cytotoxic agent and (b) said substance.

Description

DRUG THERAPY

The present invention relates to drug therapy, in particular to the treatment of tumours by localisation of cytotoxic agents at the site of the tumour.
s WO 88/07378 describes a two-component system, and therapeutic uses thereof, wherein a first component comprises an antibody fr~gment capable of binding with a tumour-associated antigen and an enzyme capable of converting a pro-drug into a cytotoxic drug, and a second 10 component which is a pro-drug which is capable of conversion to a cytotoxic drug. This general system, which is often referred to as "antibody-directed enzyme pro-drug therapy" ~ADEPT), is also described in relation to specific enzymes and pro-drugs in EP 0 302 473 and WO
91/1 1201 .
WO 89/10140 describes a modification to the system described in WO
88/07378 wherein a further component is employed in the system. This further component accelerates the clearance of the first component from the blood when the first and second components are ~lm i ~ cd 20 clinically. The second component is usually an antibody that binds to the antibody-enzyme conjugate and accelerates clearance. An antibody which was directed at the active site on the enzyme had the additional advantage of inactivating the enzyme. However, such an inactivating antibody has the lln~ irab}e potential to inactivate enzyme at the tumour sites, but its 25 penetration into tumours was obviated by the ~flclition of galactose residues to the antibody. The galactosylated antibody was rapidly removed from the blood, together with bound antibody-enzyme component, via galactose receptors in the liver. The system has been used safely and effectively in clinic~l trials. However, galactosylation of such an inactivating antibody 30 which results in its rapid clearance from blood also inhibits its penetration _ o~ norrnal tissue and inactivation of enzyme localised there.

WO 93/13805 describes a system comprising a compound comprising a target cell-specific portion, such as an antibody specific to tumour cell S antigens, and an inactivating portion, such as an enzyme, capable of converting a substance which in its native state is able to inhibit the effect of a cytotoxic agent into a substance which has less effect against said cytotoxic agent. The prolonged action of a cytotoxic agent at tumour sites is therefore possible whilst protecting normal tissues from the effects of the cytotoxic agent.

WO 93/13806 describes a further mo~lific~tion of the ADEPT systern comprising a three component kit of parts for use in a method of destroying target cells in a host. The first component comprises a target cell-specific portion and an enzym~tic~3lly active portion capable of converting a pro-drug into a cytotoxic drug; the second component is a pro-drug convertible by said enzym~tir~lly active portion to the cytotoxic drug; and the third component cornprises a portion capable of at least partly restraining the component from leaving the vascular compartment of a host when said compound is ~rlmini~tered to the v~c~ r compartment, and an inactivating portion capable of converting the cytotoxic drug into a less toxic substance.

Although all of the aforementioned methods are useful, it is still desirable to attempt to improve the specificity of the systems in order to limit side-effects to the patient.

An object of the invention is to provide a means for increasing specificity and to limit side-effects to the patient particularly in conjunction with the systems described in WO 88/07378 and WO 93/13805.

A first aspect of the invention provides a therapeutic system COlll~ g:
(a) a compound comprising a target cell-specific portion and a portion capable of converting a substance into another substance; and ~b) a molecule capable of subst~nti~lly inhibiting the conversion of said S substance, or a precursor of said molecule.

In a first particularly preferred embodiment, which is related to the system described in WO 88/07378 which is incorporated herein by reference, ~e other substance is cytotoxic and said s--~.st~n--e is sub~t~nti~lly non-10 cytotoxic, and the system further comprises said substance.

In a second particularly preferred embo-lim~nt7 which is related to the system described in WO 93/13805 which is incorporated herein by lc~el-ellce, said substance, in its native state, is able to inhibit the effect of 15 a cytotoxic agent and said other substance has less effect against said cytotoxic agent, and the system further comprises (a) a cytotoxic agent and (b) said substance.

The entity which is recognised by the target cell-specific portion may be 20 any suitable entity which is expressed by tumour cells, virally-infected cells, pathogenic microorg~ni~m~, cells introduced as part of gene therapy or normal cells of the body which one wishes to destroy for a particular reason. The entity should ~lerelably be present or accessible to the targeting portion in signific~ntly greater concentrations in or on cells 25 which are to be destroyed than in any normal tissues of the host that cannot be functionally replaced by other therapeutic means. Use of a target expressed by a cancer cell would not be precluded, for example, by its equal or greater expression on an endocrine tissue or organ. In a life-saving ~itll~tion the organ could be sacrificed provided its function was 30 either not e~ser-ti~l to life, for example in the case of the testes, or could be supplied by hormone replaceln~nt therapy. Such considerations would apply, for instance~ to the ~yroid gland, parathyroids, adrenal cortex and ovaries.

5 The entity which is recognised will o~ten be an antigen. Tumour-associated antigens, when they are expressed on the cell membrane or secreted into tumour extra-cçll~ r fluid, lend themselves to the role of targets for antibodies.

10 The term "tumour" is to be understood as r~e~ g to all forms of neoplastic cell growth, inelll~in~ tumours of the lung, liver, blood cells (le ~k~emi~), skin, pancreas, colon, prostate, uterus or breast.

The antigen-specific portion may be an entire antibody (usually, for 1~ convenience and specificity, a monoclonal antibody), a part or parts thereof (for example an Fab fr~gm~nt or F(ab')2) or a synthetic antibody or part thereof. A conjugate cO~ g only part of an antibody may be advantageous by virtue of o~ ,i..g the rate of clearance from the blood and may be less likely to undergo non-specific binding due to the Fc part.
20 Suitable monoclonal antibodies to selected antigens may be prepared by known techni~ues, for example those disclosed in ~Monoclonal Antibodies: A m~ml~l of te-~hni~lues", H. Zola (CRC Press, 1988) and i~
"Monoclonal Hybridoma Antibodies: Techniques and Applications", J.G.R. HurTell (CRC Press, 19~2). All lefelellces mentioned in ~is 25 specification are incorporated herein by reference. Bispecific antibodies may be prepared by cell filsion, by reassociation of monovalent fr~m~nts or by chemi~l cross-linking of whole antibodies, with one part of the resnltin~ bispecific antibody being directed to the cell-specific antigen and the other to the en~yme. The bispecific antibody can be ~lmini~tered bound to ~e enzyme or it can be ~ eled first, followed by the enzyme. It is ~l~r~ d that the bispecific antibodies are a~lmini~t~:red first, and after loc~li7~tion to the tumour cells, the enzyme is ~mini~tered to be captured by the tumour localized antibody. Methods for preparing bispecific antibodies are disclosed in Corvalan et al (1987) C~ancer Immunol. Immunother. 24, 127-132 and 133-137 and 138-143, and Gillsl~n-l et al (1988) Proc. Nat~. Acad. Sci. USA 85, 7719-7723.

The variable heavy (V~) and variable light (VL) domains of the antibody are involved in antigen recognition, a fact first recognised by early protease digestion experiments. Further co--ri- ~-~tion was found by "hllm~ni~tion" of rodent antibodies. Variable domains of rodent origin may be fused to constant domains of human origin such ~at the rçs~ nt antibody retains the antigenic specificity of the rodent parented antibody (Morrison et al (1984) Proc. Natl. Acad. Sci. USA 81, 6851-6855).
That antigenic specificity is conferred by variable domains and is independent of t'ne constant domains is known from experiments involving the bacterial expression of anhbody fr~gments, all cont~ining one or more variable domains. These molecules include Fab-like molecules (Better et al (1988) Science 240, 1041); Fv molecules (Skerra et al (1988) Science 240, 1038); single-chain Fv (ScFv) molecules where the VH and VL
partner domains are linked via a flexible oligopeptide (Bird et al (1988) Science 242, 423; Huston et al (1988) Proc. Natl. Acad. Sci. USA 85, 5879) and single domain antibodies (dAbs) comprising isolated V rlom~in~
(Ward et al (~989) Nature 341, 544). A general review of the t~c-hniques involved in the synthesis of anti~ody fragments which retain their specific binding sites is to be found in Winter & Milstein (1991) Nature 349, 293-299.

By "ScFv molecules" we mean molecules wherein the VH and VL partner domains are linked via a flexible oligopeptide.

The advantages of using antibody fragments, rather than whole antibodies, are several-fold. The smaller size of the fragments may lead to improved 5 pharmacological properties, such as better penetration of solid tissue.
Effector functions of whole antibodies, such as complement binding, are removed. Fab, Fv, ScFv and dAb antibody fragments can all be expressed in and secreted from E. coli, thus allowing ~e facile production of large amounts of the said fr~gments.
Whole antibodies, and F(ab')2 fr~ments are '~bivalent". By "bivalent"
we mean that the said antibodies and F(ab')2 fr~gment~ have two antigen combining sites. In contrast, Fab, Fv, ScFv and dAb fragments are monovalent, having only one antigen combining sites. Fragmentation of 15 intact immllnoglobulins to produce F(ab')2 fragments is disclosed by Harwood et al (1985) ~ur. J. Cancer Clin. Oncol. 21, 1515-1522.

IgG class antibodies are preferred.

20 Alternatively, the eutity which is recognised may or may not be antigenic but can be recognised and selectively bound to in some other way. For example, it may be a characteristic cell surface receptor such as the receptor for melanocyte-stim~ ting hormone (MSH) which is e~cpressed in high numbers in melanoma cells. The cell-specific portion may then be 25 a compound or part thereof which specifically binds to the entity in a non-immnne sense, for example as a substrate or analogue thereof for a cell-surface enzyme or as a messenger.

Considerable work has already been carried out on antibodies and 30 fragments thereof to tumour-associated aMigens and antibodies or antibody fragments directed at carcinoembryonic antigen (CEA) and antibodies or their fra~ments directed at human chorionic gonadotrophin ~hCG) can be " conjugated to carboxypeptidase G2 and the resultin~ conjugate ret~ins bo~
antigen binding and catalytic function. Following intravenous injection of S these conjugates they localise selectively in tumours expressing CEA or hCG respectively. Other antibodies are known to localise in tumours expressing the corresponding antigen. Such tumours may be primary and metastatic colorectal cancer (CEA) and choriocarcinoma (hCG~ in human patients or other forms of cancer. Al~ough such antibody-enzyme 10 conjugates may also localise in some normal tissues expressing the respective antigens, antigen expression is more diffuse in normal tissues.
Such antibody-enzyme conjugates may be bound to cell membranes via their respective antigens or trapped by antigen secreted into the illLeL~LiLial space between cells.
Examples of tumour-associated, immnne cell-associated and in~ection reagent-related antigens are given in Table 1.

TABLE 1: Cell surface antige~s for targeting a) Tumour AS~Oe;~ Antigens Antigen Antibody ~xi~tin~ uses Carcino-embryonic C46 (Amersham) Tm~3~ing and therapy Antigen 85A12 (Unipath) of colon/rectum tumours.
Placental ~lk~lin~ H17E2 (ICRF, Tm~in~ and therapy Phosphatase Travers & Bodmer) of test~ r and ovarian cancers.

Pan Carcinoma NR-LU-10 (NeoRx Tm~gin~ and therapy Corporation) of various carcin-omas inchl-ling small cell lung cancer.
Polymorphic HMFGl (Taylor- Tm~in~ and therapy Epithelial Mucin p~p~tlimitrioU, ICRF) of ovarian cancer and ~llm~n rnilk fat p~eural effusions.
globule) ,B-human Chorionic W14 Targeting of GonadoLlu~ill carboxypeptidase to human xenograft choriocarcinnm~ in nude mice (Searle et al (19gl) Br. J.
Cancer44, 137-144).
A carbohydrate on L6 ~IgG2a)' Targeting of ~k~lint?
Human Carcinomas phosphatase (Senter et al (1988) PNAS
USA 85, 4842-4846.
CD20 Antigen on B lF5 (IgG2a)2 Targeting of ~lk~lin~:
Lymphoma (normal phosphatase (Senter and neoplastic) et al (1988) PNAS
USA 85, 4842-4846.

'Hellstrom et al (1986) Cancer ~es. 46, 3917-3923 2Clarke et al (1985) Proc. Natl. Acad. Sci. USA 82, 1766-1770 Olher antigens include alphafoetoprotein, Ca-125 and prostate specific antigen.

20 b) ~mml-ne Cell Antigens Antigen Antibody h',xi~;ng uses Pan T Lymphocyte OKT-3 (Ortho) As anti-rejection Surface Antigen therapy for kidney (CD3) transplants.

-B-lymphocyte RFB4 (Janossy, Tmmnnotoxin Surface Antigen Royal Free Hospital) therapy of B cell ((~D22) lymphoma.
Pan T lymphocyte H65 (Bodmer and Tmrmlnotoxin S Surface Antigen Knowles, ICRF; treatment of acute (CD5) licensed to Xoma graft versus host Corp., USA) disease, rhPnm~toid arthritis.
c) Infectious Agent-l~el~te-l Antigens Antigen Antibody ~ ting uses Mumps virus-related Anti-mumps Antibody conjugated polyclonal antibody to diphtheria toxin ~or tre~tTn~nt of mumps.
Hepatitis B Surface Anti HBs Ag Tmmlln--toxin ~g~in~t Antigen h~a~ollla.

15 Other tumour selective targets and suitable binding moieties are shown in Table 2.

Table 2: Bindin~ moieties for tumour-selective targets and tumour-associated anti~ens Target Binding moiety Disease Tr ln~te-l EGFR anti-EGFR mAb Gl;omas Idiotypes anti-id rnAbs B-cell lymphomas EGFR (c-erbBl) EGF, TGF~ anti- Breast cancer EGFR mAb c-erbB2 mAbs Breast cancer IL-2 receptor IL-2 Lymphomas and anti-Tac mAb lellk~emi~

IL-4 receptor IL-4 Lymphomas and lenk~e~
IL-6 receptor IL-6 Lymphomas and leuk~emi~.s MSH (melanocyte- ~-MSH Melanomas sfim~ tin~ hormone) receptor Transferrin receptor Transferrin anti-TR Gliomas (TR) mAb gpgs/gp97 rnAbs Mel~nom~c p-glycoprotein cells mAbs drug-resi~t~nt cluster-1 antigen (N- mAbs Small cell lung CAM) carcinomas cluster-w4 mAbs Small cell lung carc~omas cluster-SA rnAbs Small cell lung carcin(~
cluster-6 (LeY) mAbs Small cell lung carcinomas PLAP (placental mAbs Some seminomas ~lk~line phosphatase) Some ovarian; some non small cell lung cancer CA-125 mAbs Lung, ovarian ESA (epithelial mAbs carcinoma specific antigen) CD 19, 22, 37 mAbs B-cell lymphomas 250 kDa mAbs Me}anoma proteoglycan pS5 mAbs Breast cancer TCR-IgH fusion mAbs Childhood T-cell le~lk~emi~
Blood gp A antigen mAbs Gastric and colon (in B or O tumours individuals) ¦IMUCin protein core ¦ mAbs ¦ Breast ca~cer ll It is preferred if the target cell-specific portion comprises an antibody or fragment or derivative ~ereof.

Conveniently the portion capable of converting a substance into another substance is an enzyme (or at least is a macromolecule which has catalytic activity and could, therefore, be a catalytic RNA molecule or a catalytic carbohydrate molecule or at least the catalytic portion of an eDzyme).
It is likely that the portion of the compound capable of converting a sl1bst~n~ e into another substance, when it is an enzym~tic~lly active portion, will be enzym~tic~lly active in isolation from the target cell-specific portion but it is necessary only for it to be enzym~tic~lly active 15 when (a) it is in combination with the target cell-specific portion and (b) the compound is attached to or adjacent to target cells.

The two portions of the compound of the first aspect of the invention may be linked together by any of the conventional ways of cross-linking 20 polypep~ides, such as those generally described in O'Sullivan et al (1979) Anal. Biochem. 100, 100-108. For example, the antibody portion may be enriched with thiol groups and the enzyme portion reacted with a bifunctional agent capable of reacting with those thiol groups, for example the N-hydroxysuccinimi~le ester of iodoacetic acid (NHIA) or N-2~ succinimidyl-3-(2-pyridyldi~io)propionate (SPDP). Amide and thioether bonds, for example achieved with m-maleimidobenzoyl-N-hydroxysuccinimi~le ester, are generally more stable in vivo than disulphide bonds.

30 It may not be necessary for a whole enzyme to be present in the compound of the first aspect of the invention but, of course, the catalytic portion must be present.

Alternatively, the compound may be produced as a fusion compound by recombinant DNA teGhniques whereby a length of DNA comprises respective regions encoding the two portions of the compound of the invention eit_er adjacent to one anotner or separated by a region encoding a linker peptide which does not destroy the desired properties of the compound. Conceivably, the two portions of the compound may overlap wholly or partly. The antibody component of the fusion must be sellted by at least one binding site. Examples of the construction of antibody (or antibody fr~mP-nt)-enzyme fusions are disclosed by Neuberger et al (1984) Nature 312, 604.

The DNA is then expressed in a suitable host to produce a polypeptide comprising the compound of this aspect o~ the invention. Thus, the DNA
encoding the polypeptide con~tit~-ting the compound of this aspect of the invention may be used in accordance with known terhni~les, a~~ .idlely modified in view of the tl?~chin~ con~in.o~l herein, to construct an expression vector, which is then used to transform an a~rol,liate host cell ~or the expression and production of the polypeptide of the invention.
Such terhniques include those disclosed in US Pa~ent Nos. 4,440,859 issued 3 April 1984 to Rutter et al, 4,530,901 issued 23 July 1985 to Wei~m~n, 4,582,800 issued 15 April 1986 to Crowl, 4,677,063 issued 30 June 1987 to Mark et al, 4,678,751 issued 7 July 1987 to Goeddel~
4,704,362 issued 3 November 1987 to Itakura et al, 4,710,463 issued 1 December 1987 to Murray, 4,757,006 issued 12 July 1988 to Toole, Jr.
et al, 4,766,075 issued 23 August 1988 to &oeddel et al and 4,810,648 issued 7 March 1989 to StaL~er, all of which are incorporated herein by 30 ~ ellce.

CA 02239203 l998-06-Ol . 13 The DNA encoding the polypeptide co..~ ..l;..g the compound of this aspect of the invention may be joined to a wide variety of other ONA
sequences for introduction into an a~ro~liate host. The cornp~nion DNA
will depend upon the nature of the host, the manner of the inkoduction of S the DNA into the host, and whether episomal m~int~n~e or integration is desired.

Generally, the DNA is inserted into an expression vector, such as a pl~mi~, in proper oriçnt~tion and correct reading frame for expression.
10 If n~cess~ry, the DNA may be linked to the a~ro~liate transcriptional and translational regulatory control nucleotide sequences recognised by the desired host, although such controls are generally available in the expression vector. The vector is then introduced into the host through standard techniques. Generally, not all of the hosts will be transformed 15 by the vector. Therefore, it will be n~ce~ry to select for transforrned host cells. One selection terhnique involves incorporating into the expression vector a DNA sequence, with any nt~cess~ry control elements, that codes for a selectable trait in the transforrned cell, such as antibiotic resis~ance. Alternatively, the gene for such selectable trait can be on 20 another vector, which is used to co-transforrn the desired host cell.

Host cells that have been transformed by the recombinant DNA of the invention are then cultured for a sufficient time and under a~lo~liate conditions known to those skilled in the art in view of the te~hing~ dis-25 closed herein to pern1it the expression of the polypeptide, which can thenbe recovered.

Many expression systems are known, including bacteria (for exarnple E.
coli and Rnc71/r/~ subtilis), yeasts (for example Saccharomyces cerevisiae), 30 ~ m-ontous fungi (for example Aspergillus), plant cells, animal cells and CA 02239203 l998-06-Ol insect cells.

The vectors include a procaryotic replicon, such as the ColEl ori, for propagation in a procaryote, even if the vector is to be used for expression 5 in other, non-procaryotic, cell types. The vectors can also include an a~)roL,liate promoter such as a procaryotic promoter capable of directing the expression (transcription and tr~n~l~ti~-n~ of the genes in a bacterial host cell, such as E. coli, transformed thelc;wilh 10 A promoter is an expression control element foImed by a DNA sequence that permits binding of RNA polymerase and transcription to occur.
Promoter sequences comp~tihle with exemplary bacterial hosts are typically provided in plasmid vectors cont~inin~ convenient restriction sites for insertion of a DNA segment of ~e present invention.
Typical procaryotic vector pl~mi-ls are pUC18, pUC19, pBR322 and pBR329 available from Biorad l~aboratories, (Richmond, CA, IJSA) and pTrc99A and pKK223-3 available from Pl.~l ~"~ia, Piscataway, NJ, USA.

20 A typical m~mm~ n cell vector plasmid is pSVL available from Pharmacia, Piscataway, NJ, USA. This vector uses the SV40 late promoter to drive expression of cloned genes, the highest level of expression being found in T antigen-producing cells, such as COS-1 cells.

25 An example of an inducible m~mm~ n expression vector is pMSG, also available from Ph~ cia. This vector uses the glucocorticoid-inducible promoter of ~e mouse m~ ry tumour virus long terminal repeat to drive expression of the cloned gene.

Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and are W O 97/20580 PCT/GB~ 3~A~

generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Pl~mi-ls pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating pl~micl~ (YIps) and incorporate the yeast selectable markers his3, trpl, leu2 and ura3. Pl~mi-ls pRS413-416 are Yeast Centromere S pl~mi~l~ (YCps).

A variety of methods have been developed to operatively link DNA to vectors via complementary cohesive termini. For instance, complementary homopolymer tracts can be added to the DNA segment to 10 be inserted to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.

Synthetic linkers cont~ining one or more restriction sites provide an 15 alternative method of joining the DNA segment to vectors. The DNA
segment, generated by endonuclease restriction digestion as described earlier, is treated with bacteriophage T4 DNA polymerase or E. coli DNA
polymerase I, enzymes that remove protruding, 3 '-single-stranded termini with their 3'-5'-exonucleolytic activities, and fill in recessed 3'-ends with 20 their polymerizing activities.

The combination of these activities therefore generates blunt-ended DNA
segments. The blunt-ended segments are then inr.~b~t~l with a large molar excess of linker molecules in the presence of an enzyme tha~ is a'ole 25 to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the products of ~e reaction are DNA segments carrying polymeric linker sequences at their ends. These DNA segments are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme 30 that produces termini compatible with those of the DNA segment.

Synthetic linkers cont~inin~ a variety of restriction endonuclease sites are commercially available from a number of sources including Illtelllatio~al Biotechnologies Inc, New Haven, CN, USA.

S A desirable way to modify the DNA encoding the polypeptide of this aspect of the invention is to use the polymerase chain re~ctil n as disclosed by Saiki et al (1988) Science 239, 487-491.

In this method the DNA to be enzym~ lly amplified is fl~nk~l by two 10 specific oligonucleotide primers which themselves become incorporated into the amplified DNA. The said specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art.

15 Exemplary genera of yeast co,lL~"l~lated to be useful in the practice of the present invention are Pichia, Saccharomyces, Kluyveromyces, Candida, Torulopsis, Hansenula, Schizosaccharomyces, Citeromyces, Pachysolen, Debaromyces, Metschunikowia, Rhodosporidium, Leucosporidium, Botryoascus,Sporidiobolus,Endomycopsis,andthelike. Plerelledgenera 20 are those selected from the group con~ tin~ of Pichia, Saccharomyces, Kluyveromyces, Yarrowia and Hansenula. Examples of Saccharomyces are Saccharomyces cerevisiae, Saccharomyces it~7licr~s and Saccharomyces rouxii. Examples of Kluyveromyces are Kluyveromyces fragilis and Kluyveromyces lactis. Examples of Hansenula are Hansenula polymorpha, 25 Hansenula anomala and Hansenula capsulata. Yarrowia lipolytica is an example of a suitable Yarrowia species.

Methods for the transformation of S. cerevisiae are taught generally in EP
251 744, EP 258 067 and WO 90/01063, all of which are incorporated 30 herein by l~re.~llce.

CA 02239203 l998-06-Ol Suitable promoters for S. cerevisiae include ~ose associated with the PGKl gene, GALl or GAL10 genes, CYC~l, P~lO5, TRPl, ADHl, ADH2, the genes for glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, triose phosphate isomerase, 5 phosphoglucose isomerase, glucokinase, c~-mating factor pheromone, a-mating factor pheromone, the PRBl promoter, the GUl~ promoter, and hybrid promoters involving hybrids of parts of 5' regulatory regions with parts of 5' regulatory regions of other promoters or with u~ eam activation sites (eg the promoter of FP-A-258 067).
The transcription telTnin~tion signal is preferably the 3' fl~nking sequence of a eukaryotic gene which contains proper signals for transcription terrnin~tiQn and polyadenylation. Suitable 3' fl~nking sequences may, for example, be those of the gene naturally linked to the expression control 15 sequence used, ie may correspond to the promoter. Alternatively, they may be different in which case the termin~tion signal of the S. cerevisiae AHDl gene is ~le~lled.

By "precursor of a molecule capable of subst~nti~lTy inhibiting the 20 conversion of said substance" we incll-~le any molecule which, when introduced into a host, such as a patient to be treated, will generate the said molecule capable of subst~nti~lly inhibiting the conversion of said substance. Example of molecules capable of subst~nti~lly inhibiting the conversion of said substance, and precursor of said molecule are given 25 below.
-In the first particularly preferred embodiment of the invention, the saidsubstance which is subst~nti~lly non-cytotoxic is conveniently a pro-drug and the other ~1-bs~nce which is cytotoxic is conveniently a cytotoxic 30 drug. Plainly, in this embodiment the portion capable of converting a substance into another sllbst~n~e in~ ln(les a portion capable of converting a pro-drug into a cytotoxic drug. Many pro-drugs, cytotoxic drugs and enzymes for converting the pro-drug into the cytotoxic drug are known (for example, in WO 88/07378; WO 91/11201; and EP 0 302 473 all S incorporated herein by referellce). Thus, it is preferred if the enzyme and pro-drug are chosen from the following combinations:

~lk~linf~ phosph~t~e useful for converting phosphate-cont~ininp: pro-drugs into free drugs, aryl snlrh~t~e useful for converting sulphate-cont~ining 10 pro-drugs into free drugs, cytosine ~le~min~e useful for converting non-toxic 5-fluorocytosine into the ~ntic~ncer drug 5-fluorouracil, proteases such as Serratia protease, thermolysin, subtilisin, carboxy-peptidases and cathepsins that are useful for converting peptide-cont~inin~ pro-drugs into free drugs, D-alanylcarboxypeptidases, useful for converting pro-drugs 15 that contain D-amino acid substituents, carbohydrate-enzymes such as ~-galactosidase and neur~mini~ e useful for converting glycosylated pro-drugs into free drugs, ~B-lact~m~e useful for converting drugs derivatized with ,~-lactams into free drugs and pçni-~.illin ~mifl~es useful for converting drugs derivatized at their amine nitrogens with phenoxyacetyl 20 or phenylacetyl groups into free drugs.

Other enzymes and pro-drugs include hydrolases, amidases, sulph~t~es, lipases, glucuroni~ es, phosph~t~es and carboxypeptidases, and pro-drugs be ~.~ared from any of the various classes of anti-tumour 25 compounds for example alkylating agents (nitrogen mustards) including cyclophosph~ k?, bis~llph~n, chlorambucil and nitrosoureas; interc~l~ting agents inclll~in3~ adriamycin and ~l~ctinomycin; spindle poisons such as vinca alkaloids; and anti-metabolites including anti-folates, anti-purines, anti-pyrimi-lin~ or hydroxyurea.

W O 97nO580 PCT/GB96/03000 Also included are cyanogenic pro-drugs such as amygdalin which produce cyanide upon action with a carbohydrate cleaving enzyme.

It is parti~ rly ylcr~lled if the portion capable of converting a pro-drug 5 into a cytotoxic drug is a carboxypeptidase, especially carboxypeptidase G2. It is also ~ier~.led that the pro-drug is a nitrogen mustard ~h~ te, more l)le~l~bly a benzoic acid nitrogen mustard glllt~m~te as described in WO 88107378. It is also ~lefe,led that the pro-drug is a nitrogen mustard gl..~ te derived from phenol or phenylen~ min~ mustard as lO described in WO 94/02450 (inventors P.J. Burke, R.J. Dowell, A.B.
Mauger and C.J. Springer). It is also ~lert;lled that the pro-drug is of the self-immolative type as described in WO 95/02420 (inventors C.J.
Springer and R. Marais~.

15 It will be appreciated that, advantageously, the first particularly ~le~lled emborlim.ont can be used in conjunction with the clearance system described in WO 89110140 or in conjunction with the restraining system of WO 93113806, both incorporated herein by re~lellce.

20 In the second particularly ~le~lled embo-liment of the invention the portion capable of converting a substance, which in its native state, is able to inhibit the effect of a cytotoxic agent to said other substance which has less effect ~g~in~t said cytotoxic agent is an inactivating portion.

25 By " inactivating" we inclll-le that the portion itself is able to inactivate the said substance, for example by converting it into an inactive form.

Preferably, the inactivating portion is an enzym~ti(~lly active portion.

30 Subst~nces which "inhibit" the effect of a cytotoxic agent are those which ~liminish to a useful extent the ability of the cytotoxic agent to destroy target cells. Preferably, the said ability is re~nce~l to subst~nti~ly zero.
Similarly, the inactivating portion will reduce such inhibition to a useful extent and will ~lefel~bly reduce it to subst~n~i~lly zero.
The inhibitor-inactivat~ng protein is ~rer~l~bly an enzyme capable of metabolising the said inhibitor to an inactive form.

The substance which in its native state is able to inhibit the effect of a cytotoxic agent may be any sufficiently non-toxic substance which may be converted into a substance which has less effect on said cytotoxic agent.
A suitable compound is folinic acid. Folinic acid reverses the biological effect of the cytotoxic agent trimetrexate, for example, which acts on the enzyme dihydrofolate reductase. Folinic acid is degll-t~m~ l and rendered inactive ~g~in~t trimetrexate by the enzyme carboxypeptidase G2 and other ~leglllt~m~ting enzymes.

The same principle may be applied to other anti-cytotoxic agent snbst~nces. For example, thymidine blocks the effect of a cytotoxic agent, such as CB3717 and ICI D1694 (Jodrell et al 1991, BJC 64, 833-8; Jones et al (1986) J. Med. Chem. 29, 468-472), which acts on the enzyme thymidylate synthetase. Hence a thyrmdine degrading enzyme (such as dihydrolhy~ le dehydrogenase, Shiotani & Weber 1981 J. Biol. Chem.
256, 219-224) or thymidine kinase (Shiotani et al (l g89) Cancer Res. 49, 1090-1094) may be used as the inactivating portion of the compound of the invention to render the thymidine ineffective ~g~in~t the cytotoxic agent.

Similar considerations relate to other agents which illle-rele with the 30 normal processes of nucleotide incorporation into DNA or RNA since CA 02239203 l998-06-Ol W O 97/20580 PCT/GB96/03~00 these are potentially reversible by the normal metabolite which in turn can be degraded by an ~~ iate enzyme targeted to tumour sites.

For instance, it has been shown that the cytotoxic effects of the widely 5 used cytotoxic S-fluorouracil (available from Roche Products Inc) can be at least partly ~ttPml~t~ by uridine (Groeningen et al ~1989) J. Natl.
Cancer Inst. 81, 157-162). It follows that conjl-g~tinn of an ~ r~ur antibody with a uridine degrading enzyme can be used in conjunction with 5-fluorouracil and uridine. Such a combination would be particularly 10 relevant in colorectal and breast carcinoma for which 5-fluorouracil is one of the most effective cytotoxic agents. Such a combination of agents may be further combined with folinic acid which augments the cytotoxicity of 5-fluorouracil or additionally with thymidine and a thymidine inactivating enzyme.
The inactivating portion of the compound will be chosen by reference to the anti-cytotoxic agent substance.

Enzymes other than carboxypeptidase G2 and its eguivalents can be used.
20 They should be speci~lc for the targeted metabolite but may be of human or non-hllm~n origin.

It may not be necessary to use a conventional enzyme. Antibodies with catalytic capacity have been developed (Tramontano et al Science 234, 2~ 1566-1570) and are known as 'abzymes' or catalytic antibodies. These have the potential advantage of being able to be hllm~ni7-o-1 to reduce their logenicity.

Enzymes der*ed from human lymphocytes and able to degrade thymidine have been disclosed. (Schiotani et al (1989) Cancer Res. 49, 1090-1094~ .

~ WO 97/20580 PCT/GB96/03000 A dihydrothymine dehydrogenase and thymidine kinase can be used in the system of the type herein disclosed for use in conjunction with inhibitors of thymidine synthetase.

Thymidine degrading and phosphorylating enzymes can be used as an additional element in anti-folate therapy as herein disclosed by bloclcing the thymidine salvage pathway. They can also be used in conjunction with uridine catalysing enzymes used with the cytotoxic drug 5-fluorouracil.

The bacterial enzymes carboxypeptidase Gl and G2 (CPGl and CPG2) degrade folates including methotrexate by cleavage of the terminal glllt~mic acid. The actions of the two enzymes are thought to be the same. The following description of ~lefe.,ed aspects of the invention refers to CPG2 but is equally applicable to CPGl and to any other enzymes acting on the same substrates, and to abzymes acting on the same substrates.

The isolation, purifit~tion and some of the properties of carboxypeptidase G2 from Pseudomonas sp. strain RS-16 have been disclosed by Sherwood et al (1984) Eur. J. Biochem. 148, 447-453. The cloning of the gene encoding the said carboxypeptidase G2, its nucleotide sequence and its expression in E. coli have been disclosed by Minton et al (1984) Gene 31, 31-38 and Minton et al (1983) J. Bacteriol. 156, 1222-1227. CP2G2 is available from the Division of Biotechnology, Centre for Applied Microbiological Research, Porton Down, Salisbury, UK.
Carboxypeptidase G1 (CPG1) is disclosed by Chabner et al (1972) Cancer Res. 32, 2114-2119.

Thus, in this ~le~ll~d embodiment it is particularly preferred if the portion capable of converting a substance, which in its native state, is able to inhibit the effect of a cytotoxic agent to said other substance which has less effect against said cytotoxic agent is a carboxypeptidase such as carboxypeptidase G2. It is also preferred if the said substance is folinic acid and if the said cytotoxic agent is trimetrexate.

In the first aspect of the i~vention (and in both particularly ~lertlled embodIments) by "a molecule capable of subst~nti~lly inhibiting the conversion of said substance" we mean a molecule which, when present with the compound comprising a target cell-specific portion and a portion capable of converting a substance into another substance, prevents to a useful extent the said conversion. The exten~ of inhibition is preferably >5%, more ~lefelably >10%, still more l,ler~ bly >50% and most ~lef~l~bly ~90%.

Preferably, when the portion capable of converting a substance into another substance is an enzyme or other macromolecule with catalytic activity the said molecule binds to the active site of the enzyme or other macromolecule.

By "active site" we incll--le any site on the enzyme or other macromolecule which influences the catalytic activity whether or not the site is the site of catalysis.

In further ~lcrcrcnce, the said molecule binds to the active site of the enzyme or other macromolecule and in still further ~ler~lc~-ce the said molecule is not exposed on the surface of the enzyme or other macromolecule.

Preferably, the molecule is a relatively small molecule and it is further ~le~e~led if the molecule has a relative molecular mass of less than 10000, more preferably less than 5000 and most ~l~,r~l~bly less than 1000.

When the portion capable of converting a substance to anotner substance is an enzyme or otner macromolecule with catalytic activity it is 5 particularly preferred if the molecule capable of subst~nti~lly inhibiting theconversion of the substance is a subst~nti~lly irreversible inhibitor. By "subst~nti~lly irreversible inhibitor" we include an inhibitor which, once bound to an enzyme or other macromolecule with catalytic activity, subst~nti~lly inhibits the catalytic activity and is umikely to become 10 unbound.

It is particularly preferred if the k~ a, of said enzyme, or other macromolecule witn catalytic activity, with respect ~o the molecule is < lOs~ cr~l~bly < ls-l, more ~lel~l~bly <O.ls-l, still more ~ ably 15 <O.Ols-' and most ~rerel~bly subst~nti~lly Os-l It is also particularly preferred if the Ki ~f said enzyme, or other macromolecule witn catalytic activity, is < lOO,~M, prerelably < l~M, more preferably < lnM and still more prefera~ly subst~nri~lly zero.
It is preferred, particularly in relation to the first particularly ~lerelled embo~im~nt7 if the molecule is not an antibody. It is also preferred if the molecule is not an antibody fr~ment derivable from an antibody by proteolytic digestion, such as a Fab fragment or F(ab')2 fr:~gment It is particularly ~lcr~led if the molecule capable of subst~nti~Tly inhibiting the conversion of said substance selectively inhibits tlle portion capable of converting a substance into another substance. In particular, it is ~rer~ d if said molecule does not inhibit an enz:yme activity which 30 is normally present in a host, such as a patient to be treated and, more W O 97/20580 PCT/G~96/'~3C~~

ef~;rably said molecule does not inhibit an enzyme activity which is normally present in the vascular space of a host, such as a patient to be treated.

S It is pl~r~lled if the molecule capable of substantially inhibiting the conversion of said substance is non-~lo~e~aceous.

If the molecule is a peptide it is ~rerelled that it comprises less than 50 amino acid residues, more ~lerel~bly less than 25 amino acid residues and 10 most preferably less than 10 amino acid residues.

It is further ~rer~lled if the said molecule is relatively stable to degradation in the host, such as a body of a p~ti~nt By "relatively stable to degradation" we mean that the molecule has a useful lifetime in the host 15 before it is destroyed by, or removed from, the host. It is particularly cfell~d if the compound is relatively stable to degradation when present in plasma.

A particularly l!lere,led molecule capable of sub~ lly inhibiting the 20 conversion of said substance is a molecule which is soluble in a~ueous solutions suitable for pharm~cel1tic~l ~tlmini~tration~ Conveniently the aqueous solution is suitable for intravenous or inL~ sc~ r ~rlmini~tration.

25 It is preferred that said molecule is subst~n~i~lly non-toxic, at least at the level that is ~mini~tered to a host, such as a patient to be treated.

It is also ~ler~lled if the compound does not bind to carriers in the blood such as albumin, haemoglobin and the like.

W O 97~0580 PCT/GB96/03000 It is also ~lefelled if the molecule is sul~ lly inr~p~hle of entering a cell in the body of a host? such as a patient to be treated.

Of course, the molecule capable of subst~nti~lly inhibiting the conversion 5 of a substance into another substance is selected by ref~ ce to the portion capable of converting said substance into said other substance.

For example, if the portion capable of converting said substance into said other s-lbst~n~e is carboxypeptidase G2 (CPG~) then said molecule is an 10 inhibitor of CPG2.

Studies with benzoic acid drugs and ~lllt~m~te pro-drugs of low relative molecular mass < 1000 infli- ~te that they penetrate tumours less well than normal tissues, probably because ~nours are poorly vascularised (P.
15 Antonin, PhD thesis, 1991, University of T ontlon). Only one normal tissue, brain, had a lower uptake of a pro-drug than tumour. To avoid activation of a pro-drug at any site other than in tumours it is desirable to inactivate resi~ l enzyme in normal tissues as well as in blood. Since an antibody-enzyme component localises in the tumours to a higher 20 concentration than in other tissues it follows that a srnall amount of inactivating agent, sufficient to inactivate enzyme in normal tissues, will only inactivate a small proportion of enzyme in the tumour, leaving sufficient enzyme there to activate a subsequently ~tlmini~tered pro-dlug.

25 Moreover, since a low molecular weight en7yme inhibitor may bind stoichiometrically to the active site of the enzyme, the total mass of the inhibitor n~ce.~ry to inactivate the enzyme will be very much less than that of the antibody-enzyme component.

30 The first enzyme system to be used for this approach to cancer therapy was carboxypeptidase G2 (CPG2), which cleaves the lt;~ al glllt~m~te from molecules which resemble folates wi~ a benzene r~g attached to a g~ " ,~t~. We have ~le~igne(l and made molecules to ;nactivate carboxypeptidase G2 (CPG2).
s We have found ~at suitable inhibitors of CPG2 include compounds with the general formula:
o 0 \\ OH
M~ R3 X--/ \
\\
R Z Y T
~ O

wherein R is selected from alkyl, haloalkyl, cycloalkyl, aryl, substit~lte-l aryl (1 to 5 substitn~nt~ selected from halogen, alkyl, alkoxy, NH2, OH, 20 NR2, COOH, CN, CONH2), heteroaryl (S or 6 membered ring Cont~ining 1 to 3 heteroatoms selected from N or S), OH, alkoxy, H. halogen, NH2, O-sugar, O-amino acid, N-sugar, N-amino acid, aminopyridine N-oxide (aminopy N+O-), alkenyl, phenyl, nitro, nitroso, carbohydrate, ~mino~cid, lipid, pteridine derivative; R2 is selected from OH, aminopyridine, 25 aminopyridine N-oxide and amide; R3 is H or CH3; M is selected from NH, CH2, O and S; T is selected from NH, CH2, O, S and PO; X is selected from S, Se, O, NH or is absent; Y is selected from O and S; and Z is a six-membered carbon ring, a five-membered carbon ring, a seven-membered carbon ring or a heterocyclic five-, six- or seven-membered 30 ring or a fused ring system consisting of 1 to 3 rings (either aryl or heteroaryl) and R is s~lbstilllte~l on the ring or ring system.

It is more ~rerelled if R is substituted furthest from X on ring Z. It is less preferred if R is substituted close to X on ring Z.

Preferably, Z is a benzene ring and R is s ~hstih-t~-l at the para or meta positions, most preferably at the para position. Substitution at the o~tho position is less preferred.

10 By "alkyl" we include and prefer C~ 20 alkyl, both straight chain and branched. C~ s alkyl is preferred.

By "h~lo~lkyl" we inrllltle and prefer C~ 20 haloalkyl, both straight chain or branched wherein the haloalkyl contains from one to a full number of 15 halogen atoms (ie perhalo). C,5 haloalkyl is preferred.

By "alkoxy" we include and prefer C~ 20 alkoxy. C,5 alkoxy is preferred.

It is preferred that if Z is a fused ring system each ring of the system is 20 a five, six or seven-membered ring.

It is ~lefelled if M is CH2 or NH; more ~ler~ bly NH.

It is ~l~relled if T is NH or CH2.
It is ~lef~ d if R is alkoxy; more l~lcr~lably methoxy.

It is ~lerelled if R2 is OH.

30 It is ~lcfelled if R3 is H.

It is l,rerelled if X is S.

Preferably, R is metlloxy, Z is a benzene ring where R is para to X, M
is NH2, T is CH2, X is S, Y is 0, R2 is 0~ and R3 is H. This ~ r~ d 5 compound is called In-l and is described in more detail in Example 1.

The structure of In-l is shown below:
o ~OH
CH30~ \b ,' HO
15 The chirality of glutamic acid or its analogue is either D or L (R or S).

By "lipid" we include any hydrocarbon chain, whether saturated or n~tm~ated, up to C~s in length. The nature of the lipid may be useful in directing the inhibitor molecule to a particular organ.
By "amino acid~ we include any natural or syntlletic amino acid. The nature of the amino acid will influence the solubility of the inhibitor molecule.

25 Suitable amino acids include (x-amino acids.

By "~ amino acid", we mean any compound having a group 30 R5--C--NH2 where R4 is the residual group of an amino acid, H

for example hydrogen, straight or br~nc~heA Cl~ alkyl (such as methyl, iso-propyl, 2-methylpropyl or 1-methylpropyl), hydroxyalkyl (such as -CH20H or l-hydroxyethyl), araLkyl (such as benzyl or 4-hydroxy-benzyl), thiolallcyl (such as -CH2SH), alkylthioalkyl (such as -CH2CH2SCH3~, acyl 5 (such as -CH2COOH or -CH2CH2COOH), ~mi~7~lkyl (such as -CH2CO.NH2 or -CH2CH2CO.NH2) or linear or cyclic, aromatic or non aromatic, nitrogen-cont~ininsJ heterocyclic groups such as the groups forming part of tryptophan, lysine, arginine or histidine; and Rs is a group --C(=O)R6 wherein R6 is ~H, or any ~linked or--N--linked 10 radical, for example--O--aLkyl, -O-alkyl~mino~lkyl, -O-alkoxyalkyl or --NH--NHR4 wherein R4 is straight or br~nr.h~l alkyl, optionally substituted by -CN or -OH, an amide group (such as -CONH2) or a hydrazine group (such as -(CH2)2NH(CH2)2OH). Examples of alkylaminoalkyl groups include CH3(CH3)NCH2CH2- and CH3(CH3)NCH2CH2NHCH2CH2-.

By "alkyl", we include branched or straight chain alkyl of up to 20 carbon atoms, ~er~lably 1-10 carbon atoms, more plefelably 1-6 or 1-4 carbon atoms.
We include all of the 20 o~-amino acids commonly found in naturally-occurring proteins and their D-isomers; less common naturally-occurring cY-amino acids found in proteins, such as 4-hydroxyproline, 5-hydroxylysille, desmosine, ~-N-methyllysine, 3-methylhi.~ti~line and 25 isodesmosine and their D-isomers; naturally-occurring amino acids not found in l~lott;ills, such as ~B-~l~nine, ~y-aminobutyric acid, homocysteine, homoserine, citrulline, ornithine, canavanine, djenkolic acid and ,B-cy~nc~ nine and their D-isomers; and di-, tri-, tetra-, penta-, oligo- or polypeptides based on these or other amino acids (providing that the am~no 30 acid joined to the anthracenyl ring is an ~ amino acid) which peptides may optionally include non-amino acid residues or side element~ such as sugar residues. Preferably, there is only a single amino acid group.

Thus, R4 may be: hydrogen; straight or br~n-h~-1 chain Cl~ alkyl (for 5 exarnple methyl~ isopropyl, isobutyl or sec-butyl); aryl-C,4-alkyl (for example benzyl, ,B-indolylmethyl, 4-hydroxybenzyl or 4-imifl~7.olylmethyl); Cl4-alkylthio-CI4-alkyl (for example methylthioethyl);
hydroxy-CI4-alkyl (for example hydroxymethyl or l-hydroxyethyl);
mercaptomethyl (for example -CH2SH); C,4 amide (for example -CH2C(O)NH2 or -CH2CH2C(O)NH2); Cl 1 alkyl carboxylate (for example -CH2C(O)OH or -CH2CH2C(O)OH); Cl~ alkylamine (for example (CH2)4NH2); and imino(CI 6)alkyl-amine (for example -(CH2)3NHC( = N~)NH2) -15 By "derivatives" of ~e amino acids, we inclll-le salts (acid or base addition), esters, amides, hydrazides and hydroxamic acids and other derivatives.

By "carbohydrate" we inrl~l-le all natural and synthetic carbohydrates 20 especially mono- and disacch~ri~les. Galactose and mannose are particularly preferred as they are suitable for targeting the inhibitor to hepatocytes.

Other suitable compounds are o _~OH

E~ N~--~OH

~ ~/\N~

=~ , . 32 and OH
0~
H N ~
N ~ N~--~S ~ ~ ~ ~ \'~

Details of the synthesis and properties of some of these C: PG2 inhibitors are given in Example 1. Particularly l-~ef~l,ed inhibitors are those shown 10 in bold in Scheme 1 in Example 1.

Inhibitors for use with other enzymes (as listed) inrlllrle:

a) Carboxypeptidase A (Haenseler et al (1992) Biochemistry 31, 214-220: Hydrolyses terminal peptide linkage adjacent to free carboxyl group. Wide specificity, maximally active with aromatic side group (Figure 6. Possible inhibitors ~or this enzyme are also given, Figure 7.) b) Glucuronidase (Mitaku et al (1994) Ann. Oncol. 5 (Suppl. 5), 76:
Sugar lactones are known to be inhibitors of this enzyme, such as D-s~rl~h~ric acid-1,4-1~ct~ ne for ~-glucuronidase.

c) ,B-T n~ e (Svensson et al (1993) Bioconj. Chem. 3, 176-181:
Clavulanic acid is a kIlown inhibitor of this enzyme. Other structures are also known sulb~et~m, thienamycin and imipenem.
The potent antibiotics in this family possess ,~ et~m~.~e inhibitory properties and thus run into thousands of derivatives. However, the above compounds are the most potent known at ~leSe~t.

It will be appreciated that salts of the inhibitor molecules form part of the lnvention.

In a further embodiment it is ~ler~ d that the molecule capable of 5 subst~nti~lly inhibiting the conversion of said substrate is provided in the form of a precursor.

Suitably, the precursor of said molecule culll~l ises said molecule in a form capable of releasing said molecule in a host, such as a patient. Thus, the 10 precursor may comprise said molecule bound to an entity through a linkage, said linkage being biodegradable ~and therefore cleavable within the host) or the precursor may comprise said molecule bound to an entity, whether or not through a linkage, said entity being biodegradable. In any case the molecule is released from the ~le1ul~or in the host, such as in the 15 patient to be treated.

The small molecule enzyme inactivating system can be further modified to encompass a biodegradable macromolecule. This embodiment has covalently attached molecules of inhibitor so that the macromolecule-20 inhibitor may not be able to inhibit the enzyme while it is attached to themacromolecule. However, on degradation at normal tissues the inhibitor is released and ~ ses so as to inhibit any enzyme in the vicinity. The characteristics of the macromolecule-inhibitor can be l~hemil~lly modified to match the location of distribution required for the therapy, thus 25 avoiding inhibition at the tumour site. An example of this type of system is shown in Figure 8 with lef~lence to human serum albumin (HSA). The conjugate can readily be made and purified. The conjugate is very water soluble and rapidly metabolised by a variety of tissues, thus biodistribution is similar to the non-specific distribution of the antibody-enzyme 30 conjugate. This macromolecule is kept in the circulation as the size is above the glomular filtration of the kidneys. The protein backbone is hydrolysed by lysosomal enzymes, or liver enzymes, and the resulting small molecule inhibitor diffuses into the cytoplasm and then into the extravascular region of the tissue, to inhibit any enzyme present. The other by-products are expected to be non-toxic as they are based on normal human serum albumin metabolism.

The macromolecule may comprise ~lol~ills, carbohydrates or synthetic polymers such as N2-Hydroxy propyl-methacrylamide (HMP~). The 10 macromolecule is chosen so that it can be degraded, preferably en~ym~tiç~lly, into small units, the inhibitor released and allowed to penetrate the vasculature around the tissue and thereby inhibit the non-specifically targeted enzyme.

15 The linkage between the inhibitor and ~e macromolecule is preferably of the amidomethylester type, this has a half-life suffilcient for our purposes (4-5 hr in plasma) or a peptide type with an amino acid sequence as a substrate for a particular degradative enzyme, such as Gly-Phe-Leu-~ly.
This sequence is degraded by lysosomal enzymes, such as cathepsins.
Suitable degradable linkers include those comprising esters, non-sterically-hindered disulphides, phosphates, amides, glycosides and thioesters.

Suitable non-degradable linl~ers include those comprising hydrocarbons, 25 ethers, thioethers, D-amino acids, L-sugars and sterically-hindered disulphides.

A further embodiment provides a non-degradable macromolecule comprising the inhibitor molecule, such as dextran, polylysine and 30 polyacrylamide which would be useful for long circulation times, and to W O 97/20580 PCT/GB96~'~3C~~

target particular organs that need to be protected, such as bone marrow, liver and central nervous tissue, this may be done by ~ iate derivatisation of the polymer such as galactosylation (liver), glucosylation ~brain) and polyethyleneglycosylation (increased water solubility for bone S marrow). The inhibitor molecule is ~tt~c.herl via a cleavable linker and can be cleaved at particular locations or at a particular rate, depending on which type of linker is used. 'rhe macromolecule may reach the tllmour but the rate of inhibitor released is insufficient to inhibit all the enzyme that has been targeted to the tumour site.
The use of inhibitors non-cleavably linked to a non-degradable carrier would be to inactivate any enzyme in circulation. These conjugates have advantages over clearance antibodies (such as SB43) by being cheaper to produce, and having a longer shelf life. The ph~rm~okinetic properties 15 of the polymer are more readily tailored for particular ~IgeLi~g purposes.
The synthesis of these conjugates involves ether, thioether and sterically hindered amide/ester bonds to polymers such as dextran, polylysine and ~l~in~tes Suitably, the inhibitor is ~tt~çhe~l using any one of the aforementioned linkers by methods known in the art.

The use of inhibitors non-cleavably linked to a non-degradable carrier are .lerelled for use in the system of the second particularly ~ r~ d embo-liment In a ~rerelled embodiment the precursor comprises a liposome and the molecule is released from the liposome within the host. Suitably, the inhibitor is trapped within the liposome on ~lmini~tration and is released within the host.

The use of liposomes as drug carriers has been described in G.
Gregoriadis (ed.) Liposomes as drug carriers: recent trends and progress, 3Ohn Wiley and Sons, Chirhester, UK, 1988 and l~rere-lces therein. A
variety of methods of m~king liposomes are available including those S described by Lichtenberg and Barenholz (1988) Meth. Biochem. Anal. 33, 337-462; Szoka and Papahadjopoulos (1978) Proc. Natl. Acad. Sci. US~
75, 4194; Mauk and Gamble (1979) Anal. Biochem. 94, 302-307;
Foressen et al (1992) Cancer Res. S2, 3255-3261; and Perez-Soler and Kbokhar (1992) C~ncer Res. 52, 6331-6347, all incorporated herein by 10 refeleLIce.

It is preferred if the liposomes are able to target particular organs.

A second aspect of the invention provides a method of destroying target 15 cells in a host, the method comprising the steps of ~mini~tering to the host (a) a compound comprising a target cell-specific portion and a portion capable of converting a subst~nt~ y non-toxic substance into another substance which is cytotoxic; (b) a molecule capable of snhst~nti~lly inhibiting the conversion of said subst~nti~lly non-toxic substance, or a 20 precursor of said molecule; and (c) the subst~nti~lly non-toxic substance.

Preferably, the compound comprising a target cell-specific portion and a portion capable of converting a subst~nti~lly non-toxic substance into another substance which is cytotoxic is ~tlmini~tered and, once there is an 25 ~pLilllulll balance between the target cell to normal cell ratio of the compound and the absolute level of compound associated with the target, the molecule capable of subst:lnti~lly inhibiting the conversion of said subst~nti~lly non-toxic sl~bst~nr-e, or a precursor of said molecule, is leled. Then the sub~ lly non-toxic substance (such as a pro-30 drug) is a(~ lc~ed~ The interval between a-lmini~tration of the target cell-specific portion and a portion capable of converting a subs~nti~lly non-toxic substance into another substance which is cytotoxic (for example, an antibody-enzyme conjugated) and the inhibitor molecule will depend on the target cell loc~ tion characteristics of the compound, but 5 typically it will be between 6 and 48 hours.

Suitably, pro-drug ~lministration commerl~es as soon as the plasma activity of enzyme and, by inference, the activity in normal tissues, is insufficient to catalyse enough pro-drug to cause toxicity. In the case of 10 carboxypeptidase G2, the enzyme activity is ~rerelably below 0.1 enzyme units/ml, more ~le~l~bly below 0.02 enzyme units/ml and most preferably zero. One enzyme unit of carboxypeptidase G2 is defined as the amount of enzyme which hydrolyses 1 ~ mol of methotrexate/min at pH 7.0 and 25~C.
Preferably, the target cell is a tumour cell.

A third aspect of the invention provides a method of treating a m~mm~l harbouring a tumour, the method comprising the steps of ~tlmini~tering to 20 the ~ l (a) a compound comprising a tumour cell-specific portion and a portion capable of converting a subst~nti~lly non-toxic substance into another sl-kst~n(~e which is cytotoxic; (b) a molecule capable of subst~nti~lly inhibiting the conversion of said sub~t~nti~lly non-toxic substance, or a precursor of said molecule; and (c) the substantially non-25 toxic substance.

Thus, in the second and third aspects of the invention the cytotoxic compound is released in relatively high concentration at the target or tumour site but not at non-tumour sites.

A fourth aspect of the invention provides a method of destroying a target cell in a host, the method comprising admini~tering to the host (a) a compound comprising a target cell-specific portion and a portion capable of converting a substance which, in its native state, is able to inhibit the 5 effect of a cytotoxic agent into another substance which has less effect against said cytotoxic agent; (b) a molecule capable of substantially inhibiting the conversion of said substance, or a precursor of said molecule; (c) a cytotoxic agent; and (d) said substance.

10 Preferably, the compound comprising a target cell-specific portion and a portion capable of converting a substance which, in its native state, is able to inhibit the effect of the cytotoxic agent into a substance which has less effect ~in~t said cytotoxic agent is ~lmini~tered and, once there is an ulll balance between the target cell to normal cell ratio of the 15 compound and the absolute level of compound associated with the target, the cytotoxic agent together with the substance capable of blocking the effect of the cytotoxic agent are ~t1mini~tered ~owever, an alternative method of ~Amini~tration would be possible. The amount of the compound of the invention circlTl~ting in the blood may be determined by 2~ me~ ring the activity of the enzymatic portion. Conveniently, the molecule capable of subst~n~i~lly inhibiting the conversion of said substance, or a precursor of said molecule, is ~-imini~tered to the patient before ~lmini~tration of the cytotoxic agent or the substance capable of blocking the effect of the cytotoxic agent.
Preferably, the ~l~sell~ invention provides a method of treating a m~mm~
harbouring a tumour. Suitably, the ~ llAl is first prepared for tumour therapy by a~lmini~tering a compound comprising a target cell-specific portion and a portion capable of converting a substance which, in its 30 native state, is able to inhibit the effect of the cytotoxic agent into a substance which has less effect ~g~in~t said cytotoxic agent and allowing the ratio of compound bound to target cells to compound not bound to target cells to reach a desired value. The method then further comprises ~flmini~tPring to the m~mm~l a cytotoxic agent and a substance which in S its native state is capable of inhibiting the effect of said cytotoxic agent from which a substance which has less effect on the cytotoxic agent can be generated by the inactivating portion of the said compound.
Conveniently the molecule capable of subst~nti~ly inhibiting the conversion of said substance, or a precursor of said molecule, are ~0 ~-lmini~t~red to the patient before ~tlmini~tration of the cytotoxic agent or the substance capable of blocking the effect of the cytotoxic agent.

Thus, a fifth aspect of the invention provides a method of treating a m~mm~l harbouring a tumour, the m~mm~l having been prepared for 15 treatment by ~lmini~tering a compound comprising a target cell-specific portion and a portion capable of converting a substance which, in its native state, is able to inhibit the effect of a cytotoxic agent into another substance which has less effect ~g~in~t said cytotoxic agent and allowing the ratio of compound bound to target cells to compound not bound to 20 target cells to reach a desired value, the method comprising ~lmini~tering to the m~mm~l (a) a cytotoxic agent; (b) a molecule capable of subst~nti~lly inhibiting the conversion of said substance, or a precursor of said molecule; and (c) a substance which in its native state is capable of inhibiting the effect of said cytotoxic agent from which a substance which 25 has less effect on the cytotoxic agent can be generated by the portion capable of converting a substance.

For the fourth and fifth aspects of the invention it is preferred that the molecule capable of subst~nti~lly inhibiting the conversion of said 30 substance, or a precursor of said molecule, are ~lmini~tered to the host or patient 6 to 48 hours after the ~lmini~tration of the said compound.

Thus, the fourth and fifth aspects of the invention provide a means to allow continuous action of a cytotoxic agent at target sites (such as tumour S sites) whilst protecting normal tissue from the effects of the cytotoxic agent. The substance which in its native state is capable of inhibiting the effect of the cytotoxic agent is given at a dose level sufficient to protect the normal tissues. However, the substance re~-~hinf~ tumour sites is inactivated before it can enter the cells and protect them from the 10 cytotoxic agent. In this way, normal tissues are protected from the effects of the cytotoxic agent whereas the protective molecule is rapidly degraded at tumour sites.

In the second, third, fourth and fifth aspects of the invention the 15 components can be ~lmini~tered in any suitable way, usually parenterally, for example intravenously, intraperitoneally or intravesically, in standard, sterile, non-pyrogenic formnl~ti~ ns of diluents and carriers, for example isolonic saline ~when ~-lmini~tered intravenously).

20 In the second and third aspects of the invention the pl~rclled molecules capable of subst~nti~lly inhibiting the conversion of said subst~nti~lly non-toxic substance into another substance which is cytotoxic are the same as those preferred in the first aspect of the invention an~, especially, those preferred in the first particularly preferred embo-liment.
In the fourth and fifth aspects of the invention the ~lcrclled molecules capable of subst~nti~lly inhibiting the conversion of said substance are the same as those p~erelled in the first aspect of the invention and, especially, those preferred in the second particularly l le~,lcd embodiment.

CA 02239203 l998-06-Ol When treating a host, such as a patient with a tumour, it is preierred if the ratio ~-?mini~tered, in molar terms, of the compound comprising a target cell-specific portion and a portion capable of converting a substance into another substance and the molecule capable of subst~nti~lly inhibiting the conversion of said substance is between 100:1 and 1:100, more preferably between 10:1 and 1:10, more preferably still between 5:1 and 1:5 and most preferably 1:1. In other words, it is most preferred i~ the same molar amount is ~flmini.~tered. The most suitable ratio can, however, be determined by clinician having regard to the nature of said compound and said molecule.

A sixth aspect of the invention provides a pharm~e~ti-~l composition comprising a molecule capable of subst~nti~lly inhibiting the conversion of a substance as defined in the first aspect of the invention and a pharm~cel-tically acceptable carrier.

The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in ~e art of pharmacy. Such methods include the step of bringing into association the active ingredient (compound of the invention) with the carrier which con~ti~-tes one or more accessory ingredients. In general the formulations are prepared by uniformly and intim:~tely bringing into association the active ingredient with liquid carriers.

Formulations suitable for parenteral ~lmini~tration include aqueous and non-aqueous sterile in~ection solutions which may contain anti-oxidants, l~urÇels, bacteriostats and solutes which render the fonn~ tion isotonic with the blood of the in~ended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening 30 agents. The formulations may be presented in lmit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection soIutions and suspensions may be 5 prepared from sterile powders, granules and tablets of the kind previously described.

Ple~lled unit dosage formulations are those cont~inin~ a daily dose or unit, daily sub-dose or an ~ iate fraction thereof, of an active 10 ingredient.

It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in 15 question~

It is preferred if the pharmaceutical composition comprises an inhibitor of any of the previously mentioned enzymes for use in the methods of the invention. It is particularly preferred if the enzyme is any one of 20 carboxypeptidase G2, carboxypeptidase A, glucuronidase or ,B-lact~ ce.

It is particularly preferred if the pharm~celltic~l composition comprises any one of . 43 \\ OH
M~'~ R3 X '~ ~
R Z Y T
> o wherein R is selected from alkyl, haloaLkyl, cycloalkyl, aryl, sllbsti~--t~
aryl (1 to 5 substituents selec~ed from halogen, al~yl, alkoxy, N H2, OH, N R2, COOH, CN, CONH2), heteroaryl (5 or 6 membered ring ctmt~ining 1 to 3 heteroatoms selected from N or S), OH, alkoxy, H, halogen, NH2, 15 O-sugar, O-amino acid, N-sugar, N-amino acid, aminopyridine N-oxide (aminopy N+O-), alkenyl, phenyl, nitro, nitroso, carbohydrate, ~min~ id, lipid, pteridine derivative; R2 is selected from OH, aminopyridine, aminopyridine N-oxide and amide; R3 is H or CH3; M is selected from NH, CH2, O and S; T is selected from NH, CH2, O, S and PO; X is 20 se}ected from S, Se, O, NH or is absent; Y is selected from O and S; and Z is a six-membered carbon ring, a five-membered carbon ring, a seven-membered carbon ring or a heterocyclic five-, six- or seven-membered ring or a fused ring system con~i~ting of 1 to 3 rings (either aryl or heteroaryl) and R is substituted on the ring or ring system.
- Preferred compounds are those fli~c~l~se-l above in relation to the first aspect of the invention.

A particularly ~ ed molecule is wherein R is methoxy, Z is a benzene 30 ring where R is para to X, M is NH, T is CH2, X is S, Y is O, R2 is OH
-and R3 is H.

A seventh aspect of the invention provides a compound as de~med in the sixth aspect of the invention for use in medicine.
s An eighth aspect of ~e invention provides use of a compound of the sixth aspect of the invention in the m~nl-f~r,tllre of a medicament for treating a patient with cancer.

10 Preferably, the patient has been, is being, or will be :~lmini~tered a compound as defined in the first aspect of the invention.

A ninth aspect of the invention provides a compound:

~OH
M--' 3 X ~' R- Z Y T
',.. O

2~
wherein R is selected from alkyl, haloalkyl, cycloalkyl, aryl, substituted aryl (1 to 5 substituents selected from halogen, alkyl, alkoxy, NH2, OH, NR2, COOH, CN, CONH2~, heteroaryl (5 or 6 membered ring con~ining 1 to 3 heteroatoms selected from N or S), OH, aL~coxy, H, halogen, NH2, 30 O-sugar, O-amino acid, N-sugar, N-amino acid, aminopyridine N-oxide (aminopy N+O-), aL~enyl, phenyl, nitro, nitroso, carbohydrate, zlmin~cid~
lipid, pteridine derivative; R2 is selected from OH, aminopyridine, aminopyridine N-oxide and amide; R3 is H or CH3; M is selected from NH, CH2, O and S; T is selected from NH, CH2, O, S and PO; X is 5 selected from S, Se, O, NH or is absent; Y is selected from O and S; and Z is a six-membered carbon ring, a five-membered carbon ring, a seven-membered carbon ring or a heterocyclic five-, six- or seven-membered ring or a fused ring system con~i~ting of 1 to 3 rings (either aryl or heteroaryl) and R is substituted on the ring or ring system.
Pl~r~l-ed compounds are those discussed above in relation to the first aspect of the invention.

Preferably, R is methoxy, Z is a benzene ring where R is para to X, M
15 is NH, T is CH2, X is S, Y is O, R2 is OH and R3 is H.

Synthetic methods for producing these molecules is given in Example 1.
At least some of the molecules are inhibitors of carboxypeptidase G2 thus a tenth aspect of the invention provides a method of inhibiting 20 carboxypeptidase G2 comprising providing a compound according to the ninth aspect of the invention.

The invention will now be described in more detail with reference to the following Examples and Figures wherein Figure 1 shows the in vitro cytotoxicity of In-1 on LS1747 cells.

Figure 2 shows the effect of In-1 on CPG2 enzyme activity.

30 Figure 3 shows a Lineweaver-Burke plot which in-lic~tes that In-1 is a W O 97/20~80 PCT/GB96/03000 non-competitive inhibitor. Vm~,~ has been reduced but Km is similar to the substrate, methotrexate (MTX). This intlic~tes that increasing concentration of MTX would not displace the inhibitor. More i nportantly, during therapy high dose of pro-drug should not displace the inhibitor from inhibited enzyme.

Figure 4 shows a synthetic scheme for an inhibitor for CPG2 In-1.

Figure 5 shows in general scheme for synthesis of inhibitors for CPG2.
Figure 6 shows the structure of a substrate for carboxypeptidase A.

Figure 7 shows the structures of possible inhibitors of carboxypeptidase A: these are inhibitors because of the sulphur substituents in compounds 140 and 142, and because of t'ne carbocyclic structure in place of -NH- in compound 141.

Figure 8 describes macromolecule-supported inhibitors on biologically cleavable systems. HSA is human serum albumin.
FY~mrle 1: Inhibitors of carbo~ypePtidase G2 (C~PG2) The synthesis of inhibitors for CPG2 was based on the knowledge of compounds ~or activation by CPG2 and tneir KM and kG,t values as substrates for CP~2. It was known that the enzyme required zinc2+ for its activity thus, preferably, a sulphur atom is present in the inhibitor, the said s~-lphl7t atom having a high affinity for zinc. The inhibitors in general are irreversible inhibitors or ones wit'n such a low k~at that it would allow non-specifically targeted e71zyme to be inhibited and cleared before toxic levels of active drug were generated. Conveniently, the inhibitors W O 97/20580 PCTtGB96/03000 are active site inhibitors of CPG2 which bind to the catalytic site. It is known that a glutamic acid moiety is present on the best substrates of CPG2, however, ~mm~ substituted derivatives were also found to be substrates (such as pyridyl derivatives). The phenyl ring is also desirable 5 for this particular enzyme. There is considerable flexibility in the para position of the phenyl ring (compare methotrexate structure) but only some meta and ortho substitutions on the benzene ring may be accepta~le. The linkage between the phenyl ring and the glllt~miG acid is very important as this position is at the active site. Thus, preferred inhibitors have 10 particular characteristics, quite distinct from pro-drugs. The inhibitor should have a low Kj and should remain bound in the active site resulting in a kat that is as low as possible, ideally zero as for an irreversible inhibitor. A thiocarbamate linkage s~ti~fi~s these requirements, since this linkage resulted in lower k~l values compared to benzoic acid mustard pro-drugs(Springer etal (1991) Er~r. J. Cancer27, 1361-1366). Aseries of potential compounds was designed to investigate our hypothesis, these are given in scheme (1) and Figure 5. The chemical synthesis of In-l(No 1 in Scheme 1) is given in scheme 2 and Figure 4. This derivative was chosen on the basis that the sulphur atom was present in the molecule and 20 this would complex with the zinc ion in the active site of the enzyme.
The p-methoxy-benzene thiol moiety was chosen as this did not require protection, fewer by-products would result, the product (23), scheme (2), would be lipophilic and easily purified by chromatography. The compound (1) was also a good model to quickly check the efficiency of 25 the chemistry for the synthesis of thiocarbamate derivatives. Two derivatives were successfully synthesised: the p-hydroxy-thiophenol (2) and the p-methoxy-thiophenol (1). These derivatives were used to determine Ki and 4 values with CPG2, IC50 towards LS174T cells and to test the inhibitor/ADEPT hypothesis in vivo. Data is reported here for 30 ~1). The inhibitors presented in scheme 1, include the optical isomers WO 97/20580 PCT/GB96/030~0 ~D/L) of the amino acid (glutamic acid) and structural isomers of valious substitutions on the aromatic ring. In addition, glutamic acid analogues and c~!-me~yl glutamic acid analogues are also included. Some -y-derivatised glutamic acid analogues are also given as examples.
s Scheme 1 o OH
0 NH--~
XJ~ ,>
R Z Y T
', o No R D/L ~2 T X Y Z
OMe L OH CH2 S O C

6 O-Sugar OH CH2 S O C
7 O-Aminoacid OH CH2 S O C
8 N-Sugar OH CH2 S O C
9 N-Aminoacid OH CH2 S O C
10 OMe D/L OH CH~ O S C

16 O-Sugar OH CHz O S C
17 O-~mino~ri~l OH CH2 0 S C
18 N-Sugar OH CH2 O S C
19 N-~mino~ l OH CH2 0 S C
20 OMe D/L OH CH2 - S C

24 NH2 D/L OH CHz - S C
25 O-Sugar OH CH2 - S C
26 O-~mino~(id OH CH2 - S C
27 N-Sugar OH CH2 - S C
28 N-~mino~ri-l OH CH2 - S C
29 OMe D/L OH O S O C

34 O-Sugar OH O S O C
35 O-~min~ i(1 OH O S O C
36 N-Sugar OH O S O C
37 N-~mino~cid OH O S O C
38 OMe D/L OH S S S C
39 H a~ o~yridine (AP) CH2 S O C
40 OMe D/L AP CH2 S O C
41 OMe D/L AP O S O C
42 OMe aminopyN+O~(APO~ CH2 S O C
43 APO D/L OH CH2 O S Het 44 APO D/I, APO CH2 S O Het 45 APO D/L APO O S O Het 46 O-Sugar OH CH2 S O Het 47 O-~mino~ OH CH2 S O Het 48 N-Sugar OH CH2 S O Het 49 N-Aminoacid OH CH2 S O Het S0 O-Sugar AP CH2 S O Het 51 O-~min-~cid AP CH2 S O Het 52 N-Sugar AP CH2 S O Het 53 N-Aminoacid AP CH2 S O Het 54 OMe D/L OH CH2 S O C5 55 OH D/~ OH CH2 S O C5 !;8 NH2 D/L OH CH2 S O C5 59 O-Sugar OH CH2 S O C5 60 O-~m;no~ -' OH CH2 S O C5 61 N-Sugar OH C~I2 S O C5 62 N-Aminoacid OH CH2 S O C5 63 OMe L OEI CH2 S O C7 6~ H D/L OH CHz S O C7 67 NH2 D/l, OH CH2 S O C7 68 O-Sugar OH CH2 S O C7 69 O-Aminoacid OH CH2 S O C7 70 N-Sugar OH CH2 S O C7 71 N-Aminoacid OH CH2 S O C7 72 OMe L amide CH2 S O C
73 OH L amide CH2 S O C
74 H D/L amide CH2 S O C
7~ I DIL amide CH2 S O C
76 NH2 D/L amide CH2 S O C
77 O-Sugar amide CH2 S O C
78 O-~mir~ l amide CH2 S O C
79 N-Sugar arnide CH2 S O C

80 N-~mino~ l amide CH2 S O C
81 OMe D/L OH CH2 - S HET
~ 82 OH D/L OH CH~ - S HET
83 H D/L OH CH2 - S ~IET

86 O-Sugar OH CH2 - S HET
87 O-Amin~ OH CH2 - S HET
88 N-Sugar OH CH2 - S HET
89 N-~minn~ l OH CH2 - S HET
90 OMe D/L OH O S O HET

92 H D/L OH O S O H3~T

95 O-Sugar OH O S O HET
96 O-~mino:~ri~l OH O S O HET
97 N-Sugar OH O S O HET
98 N-~Amino~ i OH O S O HET
99 alkoxy OH CH2~R/S) S O C~
100 all~enyl OH CH2(R/S) S O C~
101 Phenyl OH CH2(R/S) S O C~
102 Halogen OH CH2(R/S) S O C~
103 Nitrol~itroso OH CH2(R/S) S O C~
104 Carbohydrate OH CH2(R/S) S O G~
105.~min~ OH C~2 S O C~
106 Lipid OH CH2 S O C~
107 Pteridine d~.;v~live OH CH2 S O C~
108 OMe L OH CH2 Se O C~
109 OH L OH CH2 Se O C~
110 H D/L OH CH2 Se O C~
111 I D/L OH CH2 Se O

CA 02239203 l998-06-0l 112 N H2 D/L OH CH2 Se o 113 O-Sugar OH CH2 Se OC~
114 O-Aminoacid OH CH2 Se O OEr 11~ N-Sugar OH CH2 Se Oc~r 116 N-Aminoacid OH CH2 Se O C~
117 OMe D/L~-methyl~OH CH2 S O ~r 118 OH D/L(~-methyl)OH CH2 S O C;~
119 H D/L(c~-methyl)OH CH2 S O GHE~
120 I D/L(~-methyl)OH CH2 S O C~
10 121 rnH2 D/L(~-methyl)O H C~2 S O CnHEr 122 O-Sugar(~x-methyl) OH CH2 S Oc~:r 123 O-Aminoacid OH CH2 S O ~
124 N-Sugar(~-methyl) OH CH2 S O c~r 12S N-Aminoacid OH CH2 S Oc~:r OH
_ OH
H-N \ ~
NH2 , \~=0 0 N~N~N~= ~S
~N ~ N ~N CH3 H-N ~ OH
}~NlN~l'N~/

The aromatic ring size may also be changed, C5 refers to a five membered carbon ring and C7 refers to a seven membered carbon ring.
30 C refers to the carbon skeleton of benzene ring (C6), HET refers to heterocyclic ring structure relating to 5,6 and 7 membered ring systems.

The chirality of ~hlt~mic acid or its analogue is either D or L, (strictly R
or S nomenclature).

Particularly preferred inhibitors are those shown in bold.

Derivatives 107, 126 and 127 are methotrexate derivatives which would be potent CPG2 inhibitors but may be toxic as DHFR inhibitors (R~hm~n & Chaabra (1988).

Scheme 2 Chemistry Di-t-butyl glllt~mic acid HCl (128) (2.05g, 6.95mmol) was activated with p-nitro phenyl chlol~Ço~ ate (129) (1.4g, 6.95mmol) in dichlorom~th~n~
(20ml) in the presence of triethylamine (2ml). The reaction mixture was refluxed for 25min and then stirred for lh at room temperature The reaction mixture was flll~h~-l with argon and then a solution of p-methoxy-~enzene thiol (131) (1.08g, 7.70mmol) in dichloromethane (20ml) was added, and heated to reflux for lOmin. The cooled solution was then stirred for Shs at room temperature, monitoring by TLC for disappearance of starting material. The precipitate was filtered and the filtrate concentrated in vacuo, and chromatographed on silica gel, eluent dichloromethane. The colourless oil (132) (2.8g, 95% yield) was treated with hexane and HCI (g) and stirred overnight. The white product was filtered, washed with hexane and dried in vacuo to result in 1.16g, 53%
overall yield, mpt 115~C, of analytically pure In-l (1), Scheme (2). 'NM~
~ (D6-DMSO) ~/ppm :-1.6 (m, 2H), 2.1 (m, 2H), 3.5 (s, 3H), 3.9 (m, lH), 6.7 (d, 2H), 7.1 (d, 2H), 8.2 (d, lH): % CHN analysis requires 49.83, 4.82, 4.47 found 49.5, 4.85, 4.42. Biological data:- IC50 = 122,uM ( lh exposure, LS174T cells, Figure 1), 95% inhibition of enzyme activity =
15 ~bM (Figure 2), K; = 0.3 ~M, Line Weaver-~3urke Plot is shown in Figure 3.

A general synthesis of CPG2 inhibitors is shown in Figure 5. The structures in Scheme 1 can all be made by the route shown. The starting 5 material (133) is synthesised by standard peptide chemistrv and activated with (134). The derivatives (135) or (136) may be tethered on a polymer support, cont~inin~ a labile linker, and combinatorial chemistry carried out to produce many hundreds of derivatives at each end of the molecule.

10 A number of other potential inhibitors of carboxypeptidase G2 have been synthesised in an analogous way to IN-l ~y substitl1~in~ 4-methoxybenzenethiol with ~e appropriate thiol de~ivative and according to the scheme shown in Figure 4. Analytical data is given below.

15 (a) 4-bromophenylsulfamyl-L-~lutamic acid Mpt 142~C. CHN calc~ t~ l for Cl2 Hl2NO5SBr7 C 39.79, H 3.32, N
3.87: found C 39.76, H 3.38, N 3.77.

(b) 4-chlorophenyl~l1lf~myl-L-~lutamic acid Mpt 127~C. CHN calc~ t~-l for Cl2Hl2NOsSCl, C 45.36, H 3.80, N
4.41: found C 45.59, H 3.96, N 4.31.

(c) 4-methylphenylsulfamvl-L-~lutamic acid Mpt 129~C. CHN calc~ tecl for Cl3H,sNOsSCl, C 52.52, H 5.08, N
4.71: found C 52.52, E~ 5.10, N 4.71.
Biological data: K; = 1.05 ,~M.

(d) 3-methvlphenylsulfamyl-L-~lutamic acid monohvdrate Mpt 70~C. CHN calc~ tPA for Cl3Hl5NosscL C 49.52, H 5.43, N 4.44:
found C 49.57, H 5.43, N 4.40.

, ~ 55 (e) 3-aminophenyl~l~lf~myl-L-~lutamic acid hydrochloride Mpt 85~C. IH NMR dmso-d6: ~ ppm 8.8 (d,7.8 Hz, lH), 7.6-7.2 (m, 5H), 4.2 (m, lH), 2.3 (t,2H), 2.0 (m, lEI), 1.8 (m,lH).

5 (~ 4-pyridvlsulfamyl-L-~lutamic acid IH NMR dmso-d6: ~ ppm 8.4 (d,7.8E~z,lH), 8.3 (d, 7.8Hz, 2H), 7.4 (d, 7.8Hz, 2H), 4.1(m,1H), 2.4 (m,2H), 2.0 (m,lH), 1.9 (m,lH).

(g) 2-pyrimidinyl~lllf~myl-L-~ t~mic acid 'H NMR dmso-d6: ~ ppm 8.8 (d,4Hz, 2H), 7.3 (m,lH), 4.3 (m,lH), 2.3-1.6 (m,4H).

(h) 4-aminophenyl~lllf~myl-L-glutamic acid hydrochloride IH NMR dmso-d6: ~ ppm 8.3(s), 7.3(m), 7.1(m), 6.5 (d, 7,8Hz), 4.2 (m), 2.3(m), 2.1-1.7(m).

The following thiourethane potential inhibitor for CPG2 has been synthesised by the route described below.

(i) N-(4-trifluoromethylphenvlamino-thiocarbonyl)-L-~lutamic acid ~-methyl ester triethylamrnonium salt To a solution of L-gl-lt~mic acid--y-methyl ester hydrochloride (1.24 g, 7.68 mmol) in 20ml of dry dichlorom~th~n~ was added 4-trifluoromethyl phenyl isothiocyanate (1.17 ml,7.68 mmol). The reaction mixture was ~ stirred at room temperature for 10 mimltes and then treated with triethylamine (2.14 ml, 14.4 mmol). The reaction Illi~lUle was stirred for 20 hrs at room temperature and the clear orange solution was concentrated in vacuo. The residue was lliLulat~d with ethyl acetate and the white precipitate was removed by filtration. The filtrate was concentrated in vacuo and the residue was treated with di-isopropylether to give a creamy solid (1.8 g).
'H NMR dmso-d6: ~ ppm 11.0 (s,lH~, 8.5 (d,7.8Hz,lH), 8.4 (s,lH), 7.9 (d,7.8Hz,1H), 7.5 (t,7.8Hz,1H), 7.4 (d,7.8Hz,1H), 4.5 (d,4Hz,lEI), 3.6 (s,3H), 3.0 (q,7.8Hz,6H), 2.4-1.9 (m,4H), 1.2 (t, 7.8Hz, 9H).

Other analogous amino-thio-carbonyl-L-glutamic acid ~-methyl esters can be made using an analogous method by using an a,~ropliate isothiocyanate .
Example 2: BioloPical Data for IN-1 ~C50 -- 122 ~M, dele~ l for a 1 hour exposure ~g~in~t LS174T
human colon carcinoma cells. The growth curve is shown in ~igure 1.
15 The effect of IN-1 on CPG2 enzyme activity is shown in Figure 2. At an IN-l concentration of 15 ~LM approximately 95 % of the CPG2 activity has been inhibited as shown by its inability to convert the drug methotrexate to its de~lu~llylated form.

20 Pigure 3 is a Lineweaver-Burk plot which shows the effect of IN-l on CPG2 activity. The results suggest that IN-1 is exhibiting mixed non-competitive inhibition. The Ki of IN-1 was found to be 0.3 ~M.

Figure 9 shows the in vivo effect of IN- 1 on the enzyme activity of ~PG2 25 conjugated to the F(ab)2 fr~gm~nt of the anti-carcinoembryonic antigen monoclonal antibody ASB7. In this experiment athymic nude mice were lmini~t~red ca. 27 units of enzyme activity per mouse and 24 hours later some mice were ~lmini.stered either 2.0 mgfmouse or 6.0 mg/mouse of IN-l. One hour later the enzyme activities of a control group (no IN-1 30 ~flmini~tered) were then compared to the test groups. As shown in Figure ~7 9 the CPG2 activity was re~ ell considerably in the presence of IN-1.

- F,~mrle 3: Treatment of Patient (I) 5 Stage 1. Infusion of antibody-enzyme (CPG2) conjugate intravenously, typically over a 2 hour period (5-25,000 enzyme units per m2, depending on the pro-drug substrate).
Stage 2. 16-30 hours after Stage 1. Inhibitor given in solution by bolus or infilsion, intravenously over a period of 1-4 hours (probably 1-20 10 mg/patient, depending on amount of enzyme given in conjugate).
Stage 3. ~clmini~tration of pro-drug by multiple bolus injections I.V. or by continuous intravenous infusion, starting 1-24 hours after stage 2, (function of pro-drug with CMDA 1-3 grams daily for 2-5 days).

15 ~,Y~mrle 4: Treatment of patient ~II) Stage 1 and Stage 2 as Example 2.
Stage 3. Drug and drug antagonist (for example, trimetrexate and folinic acid) starting 1-24 hours after stage 2. Trimetrexate given by I.V.
20 infusion and co~ ing for 3-6 days. Folinic acid by I.V. route or by c~ r route or by mouth. Trimetrexate dose 40-100 mg/m2 per day. Folinic acid 10-40 mg/m2.

Claims (70)

1. A therapeutic system comprising:

(a) a compound comprising a target cell-specific targeting portion and a portion capable of converting a substance into another substance; and (b) a molecule capable of substantially inhibiting the conversion of said substance, or a precursor of said molecule wherein (1) said other substance is cytotoxic and said substance is substantially non-cytotoxic, the system further comprising said substance, or wherein (2) said substance, in its native state, is able to inhibit the effect of a cytotoxic agent and said other substance has less effect against said cytotoxic agent, the system further comprising (a) a cytotoxic agent and (b) said substance, and in either case (1) or (2) the molecule capable of substantially inhibiting the conversion of the substance has a relative molecular mass < 10 000, or a precursor of said molecule.

2. A system according to Claim 1 wherein said portion capable of converting a substance into another substance is an enzyme, or a macromolecule with catalytic activity.

3. A system according to Claims 1 or 2 wherein the target cell-specific targeting portion comprises an antibody or fragment or derivative thereof.

4. A system according to any one of the preceding claims wherein the molecule capable of substantially inhibiting the conversion of the substance has a relative molecular mass < 5000, or a precursor of said molecule.

5. A system according to Claim 4 wherein the molecule capable of substantially inhibiting the conversion of the substance has a relative molecular mass < 1000, or a precursor of said molecule.

6. A system according to any one of the preceding claims wherein the molecule capable of substantially inhibiting the conversion of the substance is non-proteinaceous.

7. A system according to Claim 2 wherein the molecule capable of substantially inhibiting the conversion of the substance binds to the active site of the enzyme, or macromolecule with catalytic activity.

8. A system according to Claim 2 wherein the molecule capable of substantially inhibiting the conversion of the substance binds to the active site of the enzyme, or macromolecule with catalytic activity, and is not exposed on the surface of said enzyme or macromolecule.

9. A system according to Claim 2 wherein the molecule capable of substantially inhibiting the conversion of the substance is a substantially irreversible inhibitor of said enzyme or said macromolecule.

10. A system according to any one of Claims 7 to 9 wherein the kcat of said enzyme with respect to the molecule is less than 1s-1.

11. A system according to Claim 10 wherein the kcat of said enzyme with respect to the molecule is substantially 0s-1.

12. A system according to any one of Claims 7 to 9 wherein the Ki of said enzyme with respect to the molecule is substantially zero.

13. A system according to any one of the preceding claims wherein the molecule is selective for substantially inhibiting the said conversion.

14. A system according to any one of the preceding claims wherein the molecule capable of substantially inhibiting the conversion of said substance is relatively stable to degradation in the body of a patient.

15. A system according to any one of the preceding claims wherein molecule is soluble in aqueous solutions that are suitable for pharmaceutical administration.

16. A system according to Claim 15 wherein the aqueous solution is suitable for intravenous or intramuscular administration.

17. A system according to Claim 1 part (1) wherein the portion capable of converting a substance into another substance is at least the catalytic portion of carboxypeptidase G2.

18. A system according to Claim 17 wherein the substance is a nitrogen mustard glutamate pro-drug.

19. A system according to Claim 17 or 18 wherein the molecule capable of substantially inhibiting the conversion of said substance into another substance is any one of wherein R is selected from alkyl, haloalkyl, cycloalkyl, aryl, substituted aryl (1 to 5 substituents selected from halogen, alkyl, alkoxy, NH2, OH, NR2, COOH, CN, CONH2), heteroaryl (5 or 6 membered ring containing 1 to 3 heteroatoms selected from N or S), OH, alkoxy, H, halogen, NH2, O-sugar, O-amino acid, N-sugar, N-amino acid, aminopyridine N-oxide (aminopy N+O-), alkenyl, phenyl, nitro, nitroso, carbohydrate, aminoacid, lipid, pteridine derivative; R2 is selected from OH, aminopyridine, aminopyridine N-oxide and amide; R3 is H or CH3; M is selected from NH, CH2, O and S; T is selected from NH, CH2, O, S and PO; X is selected from S, Se, O, NH or is absent; Y is selected from O and S; and Z is a six-membered carbon ring, a five-membered carbon ring, a seven-membered carbon ring or a heterocyclic five-, six- or seven-membered ring or a fused ring system consisting of 1 to 3 rings (either aryl or heteroaryl) and R
is substituted on the ring or ring system.

20. A system according to Claim 19 wherein R is substituted furthest from X on ring Z.

21. A system according to Claim 19 wherein Z is a benzene ring.

22. A system according to Claim 21 wherein R is substituted para to X.

23. A system according to Claim 19 wherein M is CH2 or NH.

24. A system according to Claim 19 wherein T is CH2 or NH.

25. A system according to Claim 19 wherein R is alkoxy, preferably methoxy.

26. A system according to Claim 19 wherein R is methoxy, Z is a benzene ring where R is para to X, M is NH, T is CH2, X is S, Y is O, R2 is OH and R3 is H.

27. A system according to Claim 1, part (2) wherein the portion capable of converting a substance which, in its native state, is able to inhibit the effect of a cytotoxic agent to said other substance which has less effect against said cytotoxic agent is at least the catalytic portion of carboxypeptidase G2.

28. A system according to Claim 27 wherein said substance is folinic acid.

29. A system according to Claim 27 or 28 wherein said cytotoxic agent is trimetrexate.

30. A system according to any one of Claims 27 to 29 wherein the molecule capable of substantially inhibiting the conversion of said substance into another substance is any one of wherein R is selected from alkyl, haloalkyl, cycloalkyl, aryl, substituted aryl (1 to 5 substituents selected from halogen, alkyl, alkoxy, NH2, OH, NR2, COOH, CN, CONH2), heteroaryl (5 or 6 membered ring containing 1 to 3 heteroatoms selected from N or S), OH, alkoxy, H, halogen, NH2, O-sugar, O-amino acid, N-sugar, N-amino acid, aminopyridine N-oxide (aminopy N+O-), alkenyl, phenyl, nitro, nitroso, carbohydrate, aminoacid, lipid, pteridine derivative; R2 is selected from OH, aminopyridine, aminopyridine N-oxide and amide; R3 is H or CH3; M is selected from NH, CH2, O and S; T is selected from NH, CH2, O, S and PO; X is selected from S, Se, O, NH or is absent; Y is selected from O and S; and Z is a six-membered carbon ring, a five-membered carbon ring, a seven-membered carbon ring or a heterocyclic five-, six- or seven-membered ring or a fused ring system consisting of 1 to 3 rings (either aryl or heteroaryl) and R
is substituted on the ring or ring system.

31. A system according to Claim 30 wherein R is substituted furthest from X on ring Z.

32. A system according to Claim 30 wherein Z is a benzene ring.

33. A system according to Claim 32 wherein R is substituted para to X.

34. A system according to Claim 30 wherein M is CH2 or NH.

35. A system according to Claim 30 wherein T is CH2 or NH.

36. A system according to Claim 30 wherein R is alkoxy, preferably methoxy.

37. A system according to Claim 30 wherein R is methoxy, Z is a benzene ring where R is para to X, M is NH, T is CH2, X is S, Y
is O, R2 is OH, and R3 is H.

38. A system according to any one of the preceding claims wherein the.
precursor of said molecule comprises said molecule in a form capable of releasing said molecule in a host such as a patient.

39. A system according to Claim 38 wherein the precursor comprises said molecule bound to an entity through a linkage, said linkage being biodegradable.

40. A system according to Claim 38 wherein the precursor comprises said molecule bound to an entity, whether or not through a linkage, said entity being biodegradable.

41. A system according to Claim 38 wherein said precursor comprises a liposome.

42. A method of destroying target cells in a host, the method comprising the steps of administering to the host (a) a compound comprising a target cell-specific targeting portion and a portion capable of converting a substantially non-toxic substance into another substance which is cytotoxic;
(b) a molecule capable of substantially inhibiting the conversion of said substantially non-toxic substance, or a precursor of said molecule; and (c) the substantially non-toxic substance.

43. A method of treating a mammal harbouring a tumour, the method comprising the steps of administering to the mammal (a) a compound comprising a tumour cell-specific targeting portion and a portion capable of converting a substantially non-toxic substance into another substance which is cytotoxic;
(b) a molecule capable of substantially inhibiting the conversion of said substantially non-toxic substance, or a precursor of said molecule; and (c) the substantially non-toxic substance.

44. A method of destroying a target cell in a host, the method comprising administering to the host (a) a compound comprising a target cell-specific targeting portion and a portion capable of converting a substance which, in its native state, is able to inhibit the effect of a cytotoxic agent into another substance which has less effect against said cytotoxic agent;
(b) a molecule capable of substantially inhibiting the conversion of said substance, or a precursor of said molecule;
(c) a cytotoxic agent; and (d) said substance.

45. A method of treating a mammal harbouring a tumour, the mammal having been prepared for treatment by administering a compound comprising a target cell-specific targeting portion and a portion capable of converting a substance which, in its native state, is able to inhibit the effect of a cytotoxic agent into another substance which has less effect against said cytotoxic agent and allowing the ratio of compound bound to target cells to compound not bound to target cells to reach a desired value, the method comprising administering to the mammal (a) a cytotoxic agent;
(b) a molecule capable of substantially inhibiting the conversion of said substance, or a precursor of said molecule; and (c) a substance which in its native state is capable of inhibiting the effect of said cytotoxic agent from which a substance which has less effect on the cytotoxic agent can be generated by the portion capable of converting a substance.

46. A method according to Claim 42 or Claim 43 wherein the amount, on a molar basis, of the compound of step (a) and molecule of step (b) administering is substantially the same.

47. A method according to Claim 44 or Claim 45 wherein the amount, on a molar basis, of said compound and molecule of step (b) administered is substantially the same.

48. A pharmaceutical composition comprising a molecule capable of substantially inhibiting the conversion of a substance as defined in any one of Claims 1 to 41 and a pharmaceutically acceptable carrier.

49. A pharmaceutical composition according to Claim 48 comprising an inhibitor of any one of carboxypeptidase G2, carboxypeptidase A, glucuronidase or .beta.-lactamase and a pharmaceutically acceptable carrier.

50. A pharmaceutical composition according to Claim 48 or 49 comprising any one of wherein R is selected from alkyl, haloalkyl, cycloalkyl, aryl, substituted aryl (1 to 5 substituents selected from halogen, alkyl, alkoxy, NH2, OH, NR2, COOH, CN, CONH2), heteroaryl (5 or 6 membered ring containing 1 to 3 heteroatoms selected from N or S), OH, alkoxy, H, halogen, NH2, O-sugar, O-amino acid, N-sugar, N-amino acid, aminopyridine N-oxide (aminopy N+O-), alkenyl, phenyl, nitro, nitroso, carbohydrate, aminoacid, lipid, pteridine derivative; R2 is selected from OH, aminopyridine, aminopyridine N-oxide and amide; R3 is H or CH3; M is selected from NH, CH2, O and S; T is selected from NH, CH2, O, S and PO; X is selected from S, Se, O, NH or is absent; Y is selected from O and S; and Z is a six-membered carbon ring, a five-membered carbon ring, a seven-membered carbon ring or a heterocyclic five-, six- or seven-membered ring or a fused ring system consisting of 1 to 3 rings (either aryl or heteroaryl) and R
is substituted on the ring or ring system.

51. A composition according to Claim 50 wherein R is substituted furthest from X on ring Z.

52. A composition according to Claim 50 wherein Z is a benzene ring.

53. A composition according to Claim 52 wherein R is substituted para to X.

54. A composition according to Claim 50 wherein M is CH2 or NH.

55. A composition according to Claim 50 wherein T is CH2 or NH.

56. A composition according to Claim 50 wherein R is alkoxy, preferably methoxy.

57. A pharmaceutical composition according to Claim 50 wherein R is methoxy; Z is a benzene ring where R is para to X, M is NH, T
is CH2, X is S, Y is O, R2 is OH and R3 is H.

58. A compound as defined in any one of Claims 48 to 57 for use in medicine.

59. Use of a compound as defined in any one of Claims 48 to 57 in the manufacture of a medicament for treating a patient with cancer.

60. Use according to Claim 59 wherein the patient has been, is being or will be administered a compound as defined in Claim 1.

61. A compound:

wherein R is selected from alkyl, haloalkyl, cycloalkyl, aryl, substituted aryl (1 to 5 substituents selected from halogen, alkyl, alkoxy, NH2, OH, NR2, COOH, CN, CONH2), heteroaryl (5 or 6 membered ring containing 1 to 3 heteroatoms selected from N or S), OH, alkoxy, H, halogen, NH2, O-sugar, O-amino acid, N-sugar, N-amino acid, aminopyridine N-oxide (aminopy N+O-), alkenyl, phenyl, nitro, nitroso, carbohydrate, aminoacid, lipid, pteridine derivative; R2 is selected from OH, aminopyridine, aminopyridine N-oxide and amide; R3 is H or CH3; M is selected from NH, CH2, O and S; T is selected from NH, CH2, O, S and PO; X is selected from S, Se, O, NH or is absent; Y is selected from O and S; and Z is a six-membered carbon ring, a five-membered carbon ring, a seven-membered carbon ring or a heterocyclic five-, six- or seven-membered ring or a fused ring system consisting of 1 to 3 rings (either aryl or heteroaryl) and R
is substituted on the ring or ring system.

62. A compound according to Claim 61 wherein R is substituted furthest from X on ring Z.

63. A compound according to Claim 61 wherein Z is a benzene ring.

64. A compound according to Claim 63 wherein R is substituted para to X.

65. A compound according to Claim 61 wherein M is CH2 or NH.

66. A compound according to Claim 61 wherein T is CH2 or NH.

67. A compound according to Claim 61 wherein R is alkoxy, preferably methoxy.

68. A compound according to Claim 61 wherein R is methoxy, Z is a benzene ring where R is para to X, M is NH, T is CH2, X is S, Y
is O, R2 is OH and R3 is H.

69. A method of inhibiting carboxypeptidase G2 comprising providing a compound according to any one of Claims 61 or 68.

70. Any novel feature or combination of features disclosed herein.