Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
2000-08-07
2004-09-21
Berch, Mark L. (Department: 1624)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Heterocyclic carbon compounds containing a hetero ring...
C514S274000, C514S457000, C514S481000, C514S489000, C514S512000, C544S102000, C544S313000, C549S399000, C558S271000, C560S161000
Reexamination Certificate
active
06794384
ABSTRACT:
The present invention concerns prodrugs whose aromatic oxidation, particularly their enzymatic aromatic hydroxylation, results in their activation by the release of a drug moiety. It particularly concerns anti-tumour prodrugs and those which are specifically activated by the hydroxylation activity of the P-450 enzyme CYP1B1.
Many conventional cytotoxic drugs are known (for example colchicine, esperimycin, taxol, daunomycin and staurosporin) which can be used for chemotherapeutic purposes. However, they typically suffer from the problem that they are generally cytotoxic and therefore may affect cells other than those which it is wished to target. This can be alleviated somewhat by using targeted drug delivery systems, for example direct injection to a site of tumorous tissue, or by e.g. binding the cytotoxic agent to antibody which specifically recognises an antigen displayed by cancerous cells. Alternatively, electromagnetic radiation may be used to cause chemical changes in an agent at a desired site in the body such that it becomes cytotoxic. However, all of these techniques have, to a greater or lesser extent, certain limitations and disadvantages.
It has been reported (Murray, G. I. et al., Jul. 15, 1997. Cancer Research, 57: 3026-3031) that the enzyme CYP1B1, a member of the cytochrome P450 family of xenobiotic metabolizing enzymes, is expressed at a high frequency in a range of human cancers including cancers of the breast, colon, lung, oesophazus, skin, lymph node, brain and testis, and that it is not detectable in normal tissues. This led to the conclusion (p. 3030, final sentence) that “. . . the expression of CYP1B1 in tumour cells provides a molecular target for the development of new anticancer drugs that could be selectively activated by the presence of CYP1B1 in tumour cells”. It was also reported (p.3030, column 1 lines 15-17) that CYP1B1 is capable of 4-hydroxylation of estradiol. No specific anticancer drugs were suggested.
The present inventors have now succeeded in creating a range of prodrugs having a “carrier” framework with a drug moiety conjugated to it (the prodrug other than the drug moiety is referred to below as “the rest of the prodrug”) which have little or no cytotoxic effect when in their normal state, but whose aromatic oxidation e.g. hydroxylation (for example by CYP1B1) results in the release of the drug moiety. With CYP1B1 as a hydroxylating enzyme, this provides for a self-targeting drugs delivery system in which a non-cytotoxic (or at least negligibly cytotoxic) compound can be administered to a patient, for example in a systemic manner, the compound then being hydroxylated at the site of tumour cells (intratumoural hydroxylation) to release the drug which acts to kill or otherwise affect the tumour cells. The fact that CYP1B1 is not expressed by normal cells means that the hydroxylation of the prodrug only occurs at the site of tumour cells and therefore only tumour cells are affected, thus providing a self-targeting drug delivery system.
The prodrugs of the present invention have the distinct advantage of being useful in the treatment of tumours at any site in the body, meaning that even tumours which have undergone metastasis (which are not normally susceptible to site-specific therapies) may be treated, as well of course as primary and secondary tumours.
The prodrugs may be designed to be activated by other oxidising agents, for example other enzymes (e.g. other members of the cytochrome P-450 family of enzymes) which cause hydroxylation of the prodrug. For example, a prodrug activated by hydroxylation by the human CYP1A1 isoform would be useful for the treatment of stomach cancer since this isoform is over expressed in this type of cancer (Murray et al., 1998, Br. J. Cancer, 77: 1040). Furthermore, if the prodrug is specifically activated by a fungal P-450 enzyme isoform then it has utility as a selective antifungal agent, and similarly a prodrug specifically activated by a bacterial P-450 enzyme isoform would have utility as a selective antibiotic agent.
CYP1B1 has not yet been fully characterised, and it is therefore possible that tumour-specific isoforms of it may exist which possess the same catalytic properties. The prodrugs of the present invention may, of course, be used with such enzymes.
In the case of cytochrome P-450 activated prodrugs, the therapeutic strategy achieved using them is referred to as SPEAR (Specific P-450 Enzyme Activated drug Release).
According to the present invention there is provided a prodrug comprising a drug moiety bound to a carrier framework, the prodrug being activated by aromatic oxidation of the carrier framework to release the drug moiety.
The prodrug may be activated by aromatic hydroxylation. It may be activated by enzymatic aromatic hydroxylation.
Other enzymatically-activated prodrugs are known, for example those which release a drug moiety as the result of cleavage by a peptidase enzyme. However, nowhere has it been previously suggested that a prodrug could be activated to release a drug moiety by enzymatic hydroxylation.
A prodrug according to the present invention may have the formula (Z):
wherein:
X=H, OH, OMe or N(CH
3
)
2
; and
n=0-6;
and:
R
1
=H, C
1-4
lower alkyl, or together with R
2
forms part of a cycloalkyl group which may be further substituted to form part of a polycyclic cycloalkyl group, or with R
2
forms part of a steroidal carbon framework;
R
2
=H, OMe, C
1-4
lower alkyl, or together with R
1
and/or R
3
forms part of a cycloalkyl, polycyclic cycloalkyl or steroidal carbon framework, or forms part of a polycyclic aromatic group by linkage to R
4
;
R
3
=H, OMe, C
1-4
lower alkyl or together with R
2
forms part of a cycloalkyl, polycyclic cycloalkyl or steroidal carbon framework; and
R
4
=H or is fused directly to the aromatic position designated by R
2
and either:
the drug moiety is derived from a drug having a free amino, hydroxyl or thiol group and which links it to the rest of the prodrug, such that A represents NH, NR (R=C
1-4
lower alkyl), O or S; or
the drug moiety is derived from a drug having a carboxylate group, an ester linkage joining it to the rest of the prodrug and A being absent.
Enzymatic hydroxylation of the prodrugs of formula (Z) results in the transfer of electrons from the site of hydroxylation (for example the aromatic 4 position—see
FIG. 1
) to the drug moiety, resulting in its release.
The prodrug may, for example, be an anti-tumour prodrug. The drug moiety may be cytotoxic or cytostatic, although of course it may be a moiety which has any other desired effect. Examples of classes of drug moiety include antimitotic agents, alkylating agents, antifolates, antimetabolites. DNA-damaging agents and enzyme inhibitors. Specific examples of possible cytotoxic drug moieties include 5-fluorouracil, colchicine, esperimycin, taxol, daunomycin, staurosporin, and nitrogen mustard. Alternatively, the drug moiety could be e.g. a fluorescent organic molecule which would be released in an intratumoural manner, aiding tumour detection by correlating specific cell fluorescence with the presence of the drug moiety and thus of the oxidising agent (e.g. CYP1B1) which caused its release.
Thus the term “drug” also extends to moieties which may be used for diagnostic purposes.
A possible nitrogen mustard is, for example, a para-hydroxy aniline mustard that is linked through the para-hydroxy group to the rest of the prodrug. In the case of nitrogen mustard prodrugs, the mustard function is itself activated only when the drug moiety is released from the prodrug. Another example of a nitrogen mustard which can be incorporated into a SPEAR prodrug is Nor-mustine, which can be linked directly through the mustard nitrogen atom. In this case the carbamate linked nor-mustine prodrug has very low toxicity, but upon enzymatic hydroxylation of the prodrug the potent cytotoxic agent Nor-mustine is released.
The olefin linkage
may have a cis- or trans-geometry. It may be acyclic or cyclic. It may form part of an aromatic or poly
Burke Michael Danny
Patterson Lawrence Hylton
Potter Gerard Andrew
Baker & Botts L.L.P
Berch Mark L.
De Montfort University
McKenzie Thomas
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