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9.15.3 Photoactivated prodrugs (Photodynamic Therapy; PDT)

There is considerable interest in the concept of Photodynamic Therapy which involves the administration of a non-toxic prodrug that can be activated selectively at the tumour site by light of a specific wavelength. This approach has been in use for a number of years for the treatment of psoriasis (PUVA treatment) using 8-methoxypsoralen (9.62). This agent

(9.62); 8-metfioxy psoralen is relatively non-toxic until exposed to UV light when it cross-links DNA at thymine sites causing distortion of the DNA helix.

This concept has now been extended to the treatment of cancer where it has been discovered that porphyrin-type molecules are selectively taken up by some tumours. Photofrin® is an example of a commercially available agent for Photodynamic Therapy. The drug is administered and time allowed for it to circulate and to be taken up by the tumour. Light is then administered locally to the tumour usually from a laser source. The use of surgical lasers with flexible optical fibres means that tumours in inaccessible places can be irradiated using endoscopy and modern techniques in "keyhole" surgery to minimise trauma to the patient. So far, tumours in the GI tract and on

the ovaries have been treated by PDT. An intense non-laser light source has recently been marketed for this type of therapy and many new types of prodrug are under development.

9.15.4 Antibody-drug conjugates

This type of therapy involves the administration of a cytotoxic agent attached to an antibody. Although, in principle, the antibody should guide the drug selectively to the tumour cells, little success has been achieved in practice. One problem with this approach is that, once bound to the tumour cell, the drug-antibody conjugate may not be internalised and so little cytotoxic affect is achieved. For example, studies with vinblastine-antibody conjugates demonstrated that although doses of the agent could be lowered thus reducing toxicity to some degree, the overall efficacy of the treatment was not a significant improvement over vinblastine treatment alone.

9.15.5 ADEPT

ADEPT is an acronym for "Antibody-Directed Enzyme Prodrug Therapy" (see also Section 7.4.3), and is an advance over the use of antibody-drug conjugates alone. This treatment is based on the idea that a prodrug is usually converted into its active form by some enzymatic process. In ADEPT, an antibody-enzyme conjugate is initially administered to the patient which leads to the localisation of enzyme at the tumour site. The prodrug is then administered so that conversion to the active cytotoxic agent is only achieved when it comes into contact with enzyme at the tumour site. Significant progress has been made with the development of this approach, despite the fact that the choice of enzyme is limited to those not generally found in the body. Glutamase and nitro reductase enzymes are presently being studied, and a number of potential prodrugs have been developed. Initial trials have produced very promising results.

9.15.6 GDEPT

Gene-Directed Enzyme Prodrug Therapy is related to ADEPT in that an enzyme localised at the tumour site releases a cytotoxic agent from its prodrug form. However, instead of delivering the enzyme to the tumour via an enzyme-antibody conjugate, it is delivered using a gene therapy approach. For example, research is presently being carried out into the use of viral vectors to introduce genes coding for specific enzymes into tumours and organ systems. This is an "enabling" technology in that it could be used to deliver any type of drug to a particular organ for any disease state including cancer.

9.16 NEW RESEARCH TOOLS

The search for new anticancer drugs has been enhanced by the development of a wide variety of new research tools. For example, advances in molecular biology allow new genes to be identified and then sequenced with great rapidity. An astonishing number of cancer-related genes such as BRCA1 and BRCA2 have already been discovered, and the list is increasing rapidly. As a result of identifying these genes, it is likely that gene targeted agents can be developed (see below). There is even a commercially available mouse strain known as Oncomouse® that has certain oncogenes incorporated into its genome. These mice will typically die of the gene-related tumour within a known period of time, and so the effect of novel therapeutic agents on life span can be evaluated.

Once cancer related genes and their corresponding proteins (i.e. receptors or enzymes) have been identified, it is possible to use X-ray crystallography and NMR techniques to elucidate the precise 3-dimensional shape of the protein both in the solid state and in aqueous solution. Computer-aided molecular modelling may then be used to design ligands that can interact with specific parts of the protein to modify its function (see Chapter 3).

Significant advances have also been made in screening technology. For example, with DNA-interactive agents it is not only possible to study the sequence-selectivity of DNA-binding using in vitro "footprinting" assays, but "in vivo footprinting" techniques have recently become available which allow individual drug binding sites in the nucleus of living cells to be identified. Progress in the use of robotics to handle multi-well plates means that "high-throughput" screening can be used to evaluate large numbers of compounds in a short period of time.

A new synthesis technology known as "Combinatorial Chemistry" has developed in parallel to take advantage of high throughput screening. With combinatorial techniques, molecular fragments are joined together in a sequentially random fashion to provide a mixture of a large number of molecules (possibly 1000s) within a short period of time. The use of a large number of fragments provides "molecular diversity" in which "libraries" of molecules of widely different 3-dimensional shapes are produced. The key to success with this technology is a "tagging" procedure in which ingenious techniques are used to ensure that each new compound has a unique and traceable "label" even though it may be in a mixture of hundreds or even thousands of other molecules. These libraries can be passed through high-throughput screens and the structure of any one compound in the mixture can be traced through the tag or by the history of the synthesis. The active molecule can then be re-synthesised on a larger scale for further evaluation.

9.17 FUTURE POSSIBILITIES

Cancer research is funded through government bodies and initiatives, charitable organisations and a number of pharmaceutical companies with strategic intents in this area. Although all researchers, whether industrial or academic based, would prefer to see funding for cancer research increased, it is often argued that the problem is a shortage of good ideas for new agents and therapies rather than a short-fall in funding. The present lack of effective drugs and treatments, given the resources that have been channelled into cancer research world-wide since President Nixon declared a "War on Cancer" in 1971, would tend to support this hypothesis. A few examples of new approaches under active development are briefly described below. The exciting new concepts of ADEPT and GDEPT have already been described above.

9.17.1 Gene targeting

Once a gene has been identified and associated with a particular disease such as cancer, it should be theoretically possible to develop agents capable of selectively targeting either the gene itself or the equivalent messenger RNA, making use of the type of interactions (i.e. hydrogen bonding) that allow nucleic acid strands to pair together. The binding of such agents should prevent transcription by mechanisms including the direct inhibition of RNA transcriptase. The same objective might be achievable through the use of small lower molecular weight ligands. Selectivity is the most important issue here, as it has been calculated that it may be necessary to actively recognise up to 15 to 20 base pairs of DNA in order to selectively target one gene in the entire human genome (approximately 100,000 genes). However, in practice, sufficient selectivity to minimise side-effects might be acceptable. A large number of oncogenes have now been identified and sequenced, and the technology to design and produce molecules with DNA-recognition properties is rapidly advancing. One overriding advantage of the antigene strategy is that inhibition of just one gene will prevent the formation of numerous copies of the mRNA transcript and the corresponding protein.

Agents that target DNA or the corresponding messenger RNA are known as antigene or antisense agents, respectively. Ribozymes are a special family of oligonucleotides that target and bind to mRNA but then induce cleavage.

9.17.1.1 Antigene (Macromolecules and small molecules)

The macromolecular antigene approach utilises oligonucleotides that interact with double-stranded DNA to form a so-called "triple helix". The oligonucleotide, which is typically 15-20 base pairs in length, lays in the major groove, held in place mainly by hydrogen bonding interactions. Although a great deal of effort has been put into this area, two major problems exist. Firstly, recognition of DNA is presently limited to runs of cytosines or thymines, which are presently insufficient to successfully target clinically-important gene sequences. Secondly, in practice, oligonucleotides are difficult to deliver to the DNA of cells. In addition to their cost, other problems include stability, poor pharmacokinetics and cell penetration, and rapid metabolism. A great deal of effort has been made to stabilise oligonucleotides towards enzymatic degradation or metabolism, and this has been achieved by chemically modifying the backbone phosphate groups. Strategies to enhance the stability of oligonucleotide-DNA complexes have also been pursued; for example, tethering oligonucleotides to intercalating moieties. Designer proteins are also being used to target gene sequences, and one group has succeeded in using phage display technology to produce a protein that can specifically target the bclabl oncogene.

In the small molecule area (e.g. molecules of <1500 in molecular weight), agents have now been produced that can recognise up to seven or more base pairs of DNA, and this is an intense area of research. The lexitropsins, the CC-1065 analogues, and the pyrrolobenzodiazepines are three well-known examples of families of DNA-interactive agents that are being developed to recognise extended sequences of DNA. Small molecules have an advantage in that they lack many of the problems associated with oligonucleotides or proteins such as poor stability and delivery.

9.17.1.2 Antisense oligonucleotides

An antisense oligonucleotide is a relatively short length of single-stranded DNA (e.g. 15-20 base pairs) with a sequence complementary to a region of the target mRNA. Hybridisation with the RNA then interferes with the process of translation. However, a similar set of problems exist to those described above with regard to delivery, stability and cost. In addition, this approach is not likely to be as efficient as the antigene strategy in which inhibition of just one copy of the gene will prevent many more copies of mRNA from being produced. More recently, a number of research groups have started to search for small molecules capable of interacting selectively with RNA.

9.17.1.3 Ribozymes

Ribozymes are similar to antisense oligonucleotides in that they target and bind to mRNA. However, once bound, they cause cleavage of the mRNA fragment. The base-pair sequence of the ribozyme allows it to adopt a hair-pin structure when associating with RNA. An enzyme known as RNase H is involved which induces RNA cleavage after ribozyme binding.

9.17.2 Oncogene-product inhibitors

Following on from the use of antigene or antisense agents, it may prove possible to develop small ligands capable of binding to oncoproteins (proteins produced from an oncogene), rendering them inactive. Once an oncoprotein has been identified and purified, its 3-dimensional structure can be obtained by either X-ray crystallography or NMR, and molecular modelling then used to help design low molecular weight antagonists. This approach can be supplemented by the use of combinatorial chemistry and high throughput screening. However, it is not likely to be as efficient as the antigene or antisense strategies where inhibition of just one copy of the gene will prevent many copies of the protein from being produced.

9.17.3 Gene therapy

Strategies for gene therapy in the treatment of cancer include the use of genetic tagging to identify fragments of a tumour remaining after standard treatments, vaccines containing vectors capable of expressing cytokines, the insertion of genes which activate drugs (e.g. GDEPT), and the insertion of tumour suppresser genes or the replacement of malfunctioning ones. Delivery of genes to the tumour site is generally achieved using viral vectors such as the adenovirus, although there may be problems with infection if the virus is not suitably attenuated. Alternatively, naked

DNA can be delivered directly to the tumour, and there is intense effort to develop delivery systems capable of this. There are over 100 clinical trials in progress at the present time, and a number of biotechnology companies are investing heavily in this area.

9.17.4 Growth factor and signalling pathway modulators

It is currently accepted that some tumour growth factors are autocrine (affect the secreting cell) or paracrine (affect adjacent cells) hormones. Autocrine growth factors have been studied in greater depth than paracrine ones, and several neuropeptides have been identified, including gastrin releasing peptide (found in small cell lung carcinoma), bradykinin, cholecystokinin, galanin, neurotensin and vasopressin. In response to this, novel therapeutic areas have arisen for targeting these molecules. Considerable effort is currently being put into the discovery and development of inhibitors of various signalling pathways associated with cell growth. For example, Substance P analogues have been shown to block the growth promoting effects of some of the neuropeptides listed above as has substance P(6—11). Many pathways are directly associated with specific oncogenes; for example, ras farnesyltransferase is an attractive target for the treatment of ras-dependent tumours, and a number of compounds have already been identified for development. Protein kinase C and the tyrosine kinases are also involved in signalling pathways. Bryostatin, a natural product obtained from a marine organism, activates protein kinase C and is currently under clinical investigation, as are the tyrophosphitins which disrupt the function of tyrosine kinases. Other approaches are also being considered; for example, monoclonal antibodies could be used to remove the growth factors produced. There is also interest in identifying agents that can selectively initiate apoptosis (programmed cell death) in tumour cells as opposed to healthy ones.

9.17.5 Resistance inhibitors

Drug resistance provides a major obstacle to cancer chemotherapy, and resistance may develop to more than one class of agent. Mechanisms include the induction of the MDR (multiple drug resistance) protein which can actively transport different classes of drug out of a cell, an increase in glutathione production which can "neutralise" alkylating agents, or an enhancement of metabolism and/or DNA repair. It is possible that a clinically-useful modification of drug resistance might be achievable through the use of "chemoenhancing" agents capable of sensitising tumour cells to a drug by depressing the resistance pathway. Alternatively, "chemoprotecting" agents may be able to protect healthy cells during chemotherapy. The former approach has been demonstrated experimentally by co-administration of verapamil (a calcium-channel blocker) which can enhance the effect of some anticancer drugs. Also, for use in radiotherapy, "radiosensitising" agents have been identified that can increase the sensitivity of tumours to radiation. There is a corresponding interest in "radioprotective" agents that can protect surrounding healthy tissue during radiotherapy treatment.

9.17.6 DNA-repair inhibitors

The efficiency of DNA-binding agents such as the nitrogen mustards and cisplatin is known to be reduced by the numerous repair systems that normally protect DNA from damage by carcinogens, radiation and viruses. DNA repair is mediated by a remarkable set of enzymes that first recognise the damage incurred and then set out to repair the lesion, usually by excising the damaged segment of DNA and re-synthesising a new one. The mechanism by which these enzymes work is an active area of research, and the first crystal structure of a repair enzyme has only just become available. There is presently interest in developing agents capable of inhibiting DNA repair as, theoretically, they could enhance the therapeutic value of the DNA-binding class of anticancer drugs. However, presumably there is an associated risk that carcinogens and other sources of DNA damage might be rendered more potent. The poly ADP ribosylation (PADPR) of damaged DNA is thought to be associated with the attraction of repair enzymes to the site, and so there has been a considerable effort to develop PADPR inhibitors. So far, a number of potential inhibitors have been identified, some based on simple benzamide analogues. One such agent is presently in clinical trials.

9.17.7 Telomerase inhibitors

Telomeres are repeat sequences of DNA found at the end of chromosomes. They are thought to be crucial for cell division during which they signify the end of a chromosome molecule. A number of telomeres are lost during each cycle of cell division, and this is thought to act as a type of "biological clock" leading to a natural cell death when the telomeres have been depleted. Over 80% of different tumour types have been shown to express telomerase and this is thought to offer an explanation for the immortality of cancer cells. Telomerase enzymes have now been purified, assays have been established, and a number of telomerase inhibitors have been identified. It is thought that telomerase inhibitors may have selective toxicity towards tumour cells as telomerase is not expressed to the same level in healthy cells.

9.17.8 Antimetastatic agents

A primary tumour is not often the direct cause of death of a patient as it can be removed by surgery or treated with radio- or chemotherapy. It is usually the secondary tumours (metastases) that lead to death as they become too dispersed throughout the body to make further treatment, particularly surgery, effective. Attention has recently been focused on the mechanism by which tumour cells move around the body, either via the blood or the lymph system, and establish themselves in new locations. There is an intense effort to develop drugs that can interfere with the metastatic process. For example, one drug in late clinical trials known as Marimastat® inhibits metalloproteinase enzymes which are thought to be important for metastases and tumour development.

9.17.9 Blood-flow modifying agents

A good blood supply is important for the growth of a primary tumour and can be even more important for the establishment of a metastatic tumour at a distant location. The enzyme angiogenin has recently been identified as important for establishing new microvasculature at the site of a metastatic tumour. The structure of angiogenin has now been established from X-ray crystallographic studies, and an effort is currently underway to design inhibitors.

9.17.10 Vaccines

Vaccination against cancer is an active area of research, with the objective of either activating or inducing a host response to tumour-associated antigens. Patients with melanoma have already been treated with a melanoma vaccine using BCG as an adjuvant; while there was evidence to suggest an immunological response in some patients, efficacy was poor overall. Tumours associated with viruses such as Hepatitis B and Human Papilloma Virus are also targets for a vaccination approach to cancer treatment or prophylaxis. With Hepatitis, a combination of hepatitis immunoglobulin and heat inactivated Hepatitis B vaccine produces greater protection than vaccination with hepatitis immunoglobulin alone. Research in the future may be directed towards avoiding tumour cell or antigen vaccines in favour of using antibodies. This eliminates the risk associated with passing on live tumour cells and also avoids the need to purity antigens.

9.17.11 Chemopreventive Agents ("Neutriceuticals")

As part of a general trend towards preventative medicine, there is growing interest in agents capable of antagonising the effects of carcinogens ingested in the diet or inhaled in the air. Various different mechanisms can be envisaged for agents of this type. For example, it should be possible to "neutralise" carcinogens in the GI tract and/or prevent their absorption. Alternatively, it may be possible to selectively enhance the metabolism of some carcinogens. Although highly attractive, one problem with chemoprevention is that clinical trials would need to be conducted over many years, as it is possible that cancer deaths in old age might be associated with carcinogens ingested in the first few decades of life. It is also difficult to devise in vitro screens for chemopreventive agents. Although it is possible to screen for compounds that enhance metabolism, there is no guarantee that faster metabolism, even for selected carcinogens, will lead to prevention of carcinogenesis. Indeed, some carcinogens are activated by metabolism. A number of lead compounds of diverse structure (i.e. flavensids, terpenes) have originated from epidemiological studies relating to diet. For example, soy beans are known to contain chemopreventive agents, as a soy-rich diet can lead to a lower incidence of bowel cancer. Broccoli, curry powder and orange peel are also known to contain chemopreventive agents, and compounds thought to be the active agents have been isolated and characterised.

9.18 ANTI-EMETICS

The role of anti-emetics in cancer chemotherapy is of great importance since many of the cytotoxic agents in clinical use cause profound nausea and vomiting. Uncontrolled vomiting may outweigh the benefits of treatment and also lead to poor patient compliance. Traditionally, drugs such as metoclopramide have been used, but with the addition of the 5HT3 antagonists such as ondansetron and granisetron, the incidence and severity of emesis has been substantially reduced.

FURTHER READING

Bishop, J.M. and Weinberg, R.A. (eds.) (1996) Molecular Oncology. New York: Scientific American, Inc.

Browne, M.J. and Thurlby, P.L. (eds.) (1996) Genomes, Molecular Biology and Drug Discovery. London: Academic Press Ltd.

Culver, K.W., Vickers, T.M., Lamsam, J.L., Walling, H.W. and Seregina, T. (1995) Gene therapy of Solid Tumours. British Medical Bulletin 51, 192-204.

Dalgleish, A.G. (1994) Viruses and Cancer. British Medical Bulletin 47, 21-46.

Dobrusin, E.M. and Fry, D.W. (1992) Protein Tyrosine Kinases and Cancer. Annual Reports in Medicinal Chemistry 27, 169-178.

Hochhauser, D. and Harris, A.L. (1991) Drug Resistance. British Medical Bulletin 47, 178-196.

Larson, E.R. and Fischer, P.H. (1989) New Approaches to Antitumour Therapy. Annual Reports in Medicinal Chemistry 24, 121-128.

Lee, M.D., Ellestead, G.A. and Borders, D.B. (1991) Calicheamicins: Discovery, Structure, Chemistry and Interaction with DNA. Accounts of Chemical Research 24, 235-243.

Lemoine, N.R. and Cooper, D.N. (eds.) (1996) Gene Therapy. Oxford: BIOS Scientific Publishers Ltd.

Macdonald, F. and Ford, C.H.J. (1997) Molecular Biology of Cancer. Oxford: BIOS Scientific Publishers Ltd.

Miller, A.D. (1992) Human Gene Therapy Comes of Age. Nature (London) 357, 455460.

Mulligan, G.C. (1993) The Basic Science of Gene Therapy. Science 260, 926-932.

Nature (Supplement to issue 6604) (1996) Intelligent Drug Design 384, 1-26.

Neidle, S.J. and Waring, M.J. (eds.) (1993) Molecular Aspects of Anticancer Drug-DNA Interactions. London: The Macmillan Press.

Pratt, W.B. and Ruddon, R.W. (eds.) (1979) The Anticancer Drugs. Oxford: Oxford University Press.

Pullman, B. (1991) Sequence Specificity in the Binding of Antitumour Anthracyclines to DNA. Anti-cancer Drug Design 7, 95-105.

Scientific American (Special Issue) (1996) What You Need to Know About Cancer 275, 4-167.

Silverman, R.B. (1992) The Organic Chemistry of Drug Design and Drug Action. London: Academic Press Inc.

Suffness, M. (1993) Taxol: From Discovery to Therapeutic Use. Annual Reports in

Medicinal Chemistry 27, 305-314. Vousden, K.H. and Farrell, P.J. (1994) Viruses and Human Cancer. British Medical Bulletin 50, 560-581.

Workman, P. (ed.) (1992) New Approaches in Cancer Pharmacology: Drug Design and Development. London: Springer-Verlag. Yarnold, J.R., Stralton, M. and McMillan, T.J. (eds.) (1996) 2nd Edition Molecular Biology for Oncologists. London: Chapman and Hall.

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