T

9.10.2 Mithramycin

Mithramycin (9.47) is a chromomycin antibiotic isolated from Streptomyces plicatus. It is believed to exert its antitumour activity by forming a complex with magnesium ions and interfering with DNA-directed RNA synthesis.

Mithramycin is clinically useful in the treatment of testicular cancers refractory to standard chemotherapy. However, it seems to have a greater role in reversing hypercalcaemia (associated with malignant disease) through its action on osteoclasts.

9.10.3 Mitotane

Mitotane (9.48) is a congener of DDT and is used in the treatment of adrenal carcinoma in which it causes tumour regression and alleviates some of the symptoms associated with excess steroid production.

f9.48); mild! anc (9.49); aiathiypiiiie j I miiraTi®)

9.11 IMMUNOSUPPRESSIVE AGENTS 9.11.1 Azathioprine

Azathioprine (9.49) is a prodrug of 6-mercaptopurine (Section 9.7.2) that contains an imidazoyl "protecting" group. The immunosuppressive effect is believed to be due to the disruption of nucleic acid metabolism at stages where cell proliferation occurs in response to antigen exposure. Azathioprine is therefore useful in the treatment of leukaemias. Allopurinol is often given concurrently to inhibit xanthine oxidase which would otherwise metabolically inactivate the released mercaptopurine.

9.11.2 Cyclosporin (Neoral®)

Cyclosporin (9.50) is an immunosuppressive agent used to facilitate the acceptance of bone marrow grafts. It can be used as a therapeutic option in the treatment of leukaemias. The drug is expensive and some therapeutic regimens include concomitant ketoconazole (an antifungal agent) which, by inhibiting the liver enzymes involved in the metabolism of cyclosporin, promotes circulating levels of the agent.

(9.SÛ); cyclosporin (Neoral®)

9.12 ANTI-HORMONAL AGENTS

It has long been recognised that tumours derived from hormone-dependent tissues are themselves dependent on the same hormone. This has been demonstrated by the remissions observed in premenopausal breast cancer following ovariectomy and in prostatic cancer following orchiectomy.

9.12.1 Breast Cancer

Oestrogens act as promoters rather than initiators of breast tumour development and can also facilitate tumour invasiveness by stimulating the production of proteases which can degrade the extracellular matrix.

Oestrogen receptors (ERs) can be detected in 60-80% of human breast cancers. They consist of specific oestrogen-binding proteins (termed oestrophilins) located in the nucleus of oestrogen responsive breast cells. Oestradiol diffuses into the nucleus, where it binds to an unoccupied receptor site to form an oestrogen-receptor complex. This complex then binds to genomic DNA and stimulates mRNA synthesis which in turn stimulates protein synthesis at the ribosome and subsequent cell division. Oestrogen receptors have been found to be related to the sensitivity of the tumour to anti-endocrine treatment in that approximately 60% of ER positive breast cancer patients respond to endocrine therapy.

9.12.1.1 Anti-oestrogens

After surgery with associated radiation therapy to remove the tumour mass, antiendocrine therapy is initiated to prevent the growth of metastases. Tamoxifen (9.51) is used as a first line anti-oestrogen; it competes with oestrogen for ER so preventing oestrogen activation and subsequent tumour growth. One third of non-selected postmenopausal patients respond and the rate is higher (50-60%) for ER-positive tumours.

Tamoxifen is very well tolerated with only a few side effects related to its weak agonist action. However, it is speculated that tamoxifen use could be associated with an increased risk of endometrial cancer. Newer antagonists with reduced oestrogenic activity e.g. alkylamide analogues of oestradiol (9.53, ICI 164, 384 and 9.54, ICI 182,780) and pyrrolidine-4-iodotamoxifen (idoxifene) have been introduced.

Tamoxifen-resistant tumours are sometimes amenable to treatment with a second line drug such as an aromatase inhibitor or occasionally a progestin (e.g. medroxyprogesterone acetate).

9.12.1.2 Aromatase inhibitors

Androstenedione and testosterone are converted by the cytochrome P450 enzyme aromatase to oestrone and oestradiol, respectively, as the final step in the steroidogenesis pathway from cholesterol. Selective inhibition of aromatase would lead to reduced oestrogen plasma levels without affecting other hormones produced by the steroidogenesis pathway.

Aminoglutethimide (AG) was the first clinically useful aromatase inhibitor to be discovered and is still used despite undesirable CNS effects and a lack of target enzyme specificity. The latter probelm is associated with effects on other cytochrome P450

(9.54); ICI 182,780; R = (CH^OtCH^CFjCF, enzymes in the pathway. Several potent and more-specific inhibitors free from CNS effects have been introduced in recent years. These include the reversible inhibitors, fadrozole, Arimidex®, letrozole, vorozole and the irreversible inhibitors, 4-hydroxyandrostendione, plomestane and exemestane. These agents are discussed in Chapter 6 and Section 8.6.3 in more detail.

9.12.2 Prostatic cancer

Prostatic cancer is promoted by the androgen dihydrotestosterone (DHT) derived from testosterone by the action of 5a-reductase. Prostatic cancer is mainly hormone-dependent and is usually well developed on presentation so that survival rates are low and treatment is aimed at increasing the time of survival and the quality of life. Surgical removal of the prostate or testes (orchidectomy) are now less prevalent treatments having been largely replaced by endocrine therapy (oestrogens) and in recent years by treatment with anti-androgen or luteinising-hormone releasing hormone (LHRH) analogues.

9.12.2.1 Oestrogen therapy

Oestrogen therapy (diethylstilboestrol, DES) acts by inhibiting the hypothalamic-pituitary system through a negative feed-back mechanism resulting in a fall in the secretion of luteinising hormone (LH) from the pituitary and a subsequent decrease in testosterone synthesis by the testes (see Figure 9.1). This "chemical castration" has now lost favour due to cardiovascular complications and the feminising side effects associated with oestrogen.

9.12.2.2 LHRH analogues

The introduction of LHRH analogues has provided an alternative to oestrogens and orchidectomy in the treatment of advanced prostate cancer but without significant side-effects. Naturally occurring LHRH (Pyr-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-

Gly-NH2) has a short half-life (and a pulsitile action on the receptor) but by substituting the amino acid at the 6th position, deleting the amino acid at the 10th position, and adding an ethylamide group to the proline residue at the 9th position, a synthetic analogue (leuprorelin, Prostop SR®) is produced that has a greatly increased potency together with prolonged activity and a non-pulsitile action on the receptor. The initial effect of LHRH agonists is stimulation of the secretion of LH and follicle stimulating hormone (FSH) leading to elevations of serum testosterone to 140-170% of basal levels within several days. Continuous administration, however, leads to dramatic inhibitory effects through a process of "down regulation" of LHRH pituitary membrane receptors, and a reduction in gonadal receptors for LH and FSH, resulting in suppression of testosterone secretion comparable to surgical castration. Thus chronic administration causes the pituitary gland to become refractory to additional stimulation by endogenous LHRH and testicular androgen production is prevented.

The most commonly used LHRH agonists are leuprolide (D-Leu6-DES-Gly-NH210-LHRH ethylamide), buserelin (D-Ser(Bu')6-DES-Gly-NH210-LHRH ethylamide, Suprefact®) and goserelin (Ser-(Bu')6-AZ-Gly-NH210-LHRH, Zoladex®). A specific side-effect of LHRH analogue treatment is a transient worsening of symptoms (including increased bone pain) during the first week of therapy as a result of the initial testosterone surge, with approximately 3-17% of patients affected. This has led to co-administration of anti-androgens before or with the first LHRH analogue to prevent this effect. Other side-effects of these agents include atrophy of the reproductive organs, loss of libido, and impotence. As they are only able to cause a decrease in testicular androgens whilst leaving adrenal androgen production unaffected, the use of anti-androgens in combination with LHRH agonists has been studied and found to give greater survival rates than LHRH agonists alone.

9.12.2.3 Anti-androgens

Anti-androgens inhibit the binding of dihydrotestosterone and other androgens to the androgen receptors in target tissues. Target cells are located in all areas of the body that depend on androgens, e.g. the male genital skin, the seminal vesicles, the prostate, fatty tissues and breast tissue as well as the hypothalamus and the pituitary. Anti-androgens bind to the androgen receptor, creating a receptor-(anti-androgen) complex which is unstable and transient. Therefore, androgen-dependent gene transcription and protein synthesis are not stimulated. However, anti-androgens are capable of blocking the tropic effect of all androgens, not only in the prostate but also in the hypothalamus and pituitary.

These agents have both central (hypothalamus/pituitary) and peripheral (prostate) effects, and the most extensively studied is cyproterone acetate (9.55, Cyprostat®). In the target prostatic cell, it acts as a competitive inhibitor of the binding of DHT to androgen receptors and centrally, it lowers LH and, therefore, plasma testosterone levels, due to its progestin-like activity. A Phase III study has revealed objective responses in 33% of patients with advanced prostate cancer and stabilisation of the disease in 40%. Lethal cardiovascular events were slightly higher with this drug than with DES.

Non-steroidal anti-androgens have limited central activity but significant and potent peripheral effects, displacing testosterone and DHT from the androgen receptor, not only in the prostate, but also at the level of the hypothalamus. This latter blocking effect leads to an increase in the release of LHRH and subsequent LH production, leading to a slow but gradual rise in serum testosterone levels to overcome the blockade which can then stimulate prostatic tumour growth.

Flutamide (9.56, Drogenil®) was the first non-steroidal anti-androgen to be developed. Flutamide is a prodrug, the active metabolite being hydroxyflutamide (9.57) which acts

by inhibiting the uptake of testosterone or the nuclear binding of testosterone and DHT to the androgen receptor. Hydroxyflutamide has peripheral and central activity on all androgen target cells, although gynaecomastia is a common side-effect with approximately 61% of patients being affected, 10% of whom suffer severely. Other side-effects include nausea, vomiting and diarrhoea. As flutamide is a pure anti-androgen, it does not inhibit gonadotrophin production by the pituitary and so gonadal and adrenocortical steroidogenesis continues unabated. This results in a normal or elevated serum testosterone level which allows patients to retain their libido.

Molecular modelling studies of hydroxyflutamide have attributed its greater binding affinity to the dominant conformation in which the NH bond is hydrogen bonded to the hydroxyl function. The related compounds, Anandron (9.58) and Casodex (9.59) (see later), have similar conformations.

Figure 9.1 Control of androgen levels by the Hypothalamus-Pituitary axis.

Nilutamide (anandron) is another non-steroidal anti-androgen that has some structural similarities to flutamide, although it does not require metabolic activation. It is well absorbed and has a much longer half-life than flutamide (45 hours, compared to 5-6 hours for flutamide), permitting a once-daily dosage. It is centrally and peripherally active, and is therefore associated with a rise in plasma LH and subsequently plasma testosterone. Nilutamide is particularly useful in patients who are intolerant to flutamide.

Casodex® is the most recent anti-androgen to be studied. It is well absorbed and has a half-life of 5-7 days. It blocks androgen receptors peripherally and centrally and is associated with increased plasma testosterone levels in men. Casodex® is currently undergoing clinical investigation as a monotherapy and also in combination with other agents for advanced prostate cancer. The results of studies to date suggest that it causes few significant side-effects.

9.13 ENZYMES

Asparaginase (crisantapase, Erwinase®) is produced by Erwinia chrysanthemi and is a 133,000 molecular weight tetrameric protein used in the treatment of acute lymphoblastic leukaemia. The mechanism of action is based on the fact that these particular tumour cells have very low levels of asparagine which is required for cell growth; instead they must obtain this amino acid exogenously. Healthy cells, on the other hand, can synthesis their own asparagine. The strategy involves the administration of crisantapase which reduces the concentration of asparagine in the body by converting it to aspartic acid and ammonia, thereby removing asparagine from the protein synthesis cycle. Whereas healthy cells can rapidly synthesise their own supply of asparagine, the tumour cells succumb to the reduced levels in their environment. Resistance to the drug develops when the tumour cells begin to synthesise their own asparagine.

9.14 BIOLOGICALS

9.14.1 Interferon alpha (Intron A®, Roferon A®, Walferon®)

Interferon was discovered in 1957 by scientists at the National Institute for Medical Research (UK). It is a glycoprotein induced in response to viral infections and is usually effective only in species in which it is produced. It was first investigated as an antiviral agent and shown to have a broad spectrum of activity. Most interferons produce an 80-100% reduction in the incidence of experimentally-induced common colds, and their use is also being examined further in treating chronic hepatitis B, papilloma viruses (warts) and virus infections associated with immunosuppressed patients following renal transplantation.

A new system of nomenclature for the interferons has been devised. To qualify as an interferon a substance must be a protein which exerts virus non-specific anti-viral activity in homologous cells through cellular metabolic processes involving synthesis of both RNA and protein. The preferred abbreviation for interferon is IFN. Each interferon is then designated by the animal of origin, e.g. human: HuIFN, murine: MuIFN, bovine: BovIFN. The interferons are next classified into types according to antigen specificities, e.g. a, p and y, which correspond to the previous designations of leucocyte, fibroblast and type II immune, respectively. It was thought that previous type names were misnomers as both leucocytes and fibroblasts can produce each of the two types (a and p) of interferon. a and p Interferons are usually stable in acid media whilst y interferons are acid-labile. Properly documented differences in molecular size appear to be useful parameters for characterization until more stringent criteria such as amino acid sequence or monoclonal antibody recognition are forthcoming. Molecular weight designations are indicated as HuIFN-a (18 K), MuIFN-P (39 K), etc.

Interest in the potential anticancer activity of interferon first arose following encouraging results obtained in Sweden while treating osteogenic sarcoma and myelomatosis. In 1981 doctors in Yugoslavia, using human leucocyte interferon preparations injected directly into tumours, reported substantial improvements or total remissions in cancers of the head and neck. Although the mechanism of antitumour activity is unknown, either a direct effect on malignant cells or a stimulation of the host's immune system have been postulated. Despite the intense interest generated by the interferons when first discovered, a considerable time elapsed before the compounds were brought into clinical use, the major difficulty being commercial production of sufficient quantities. Large doses are required for treatment, and initially only minute amounts of varying levels of purity became available from human tissue culture methods. However, from the early 1980s there were significant advances in production techniques, including the development of recombinant DNA technology, which allowed biosynthetic interferons of high purity to be made available in clinically-useful quantities.

The three principal types of interferon, namely a-IFN produced by leucocytes and other lymphoid (lymphoblastoid) cells, P-IFN from fibroblasts and y- (or immune) IFN became available initially. More recently, lymphoblastoid interferon, Wellferon (a complex mixture of a-interferons and recombinant interferon) and Intron A (a pure ainterferon) have become available. a-Interferon is now licensed for the treatment of Kaposi's sarcoma, and certain blood cancers.

Combination chemotherapy with interferon appears to be promising with some treatment regimes. For example, combinations of Intron A® with doxorubicin, cisplatin, vinblastine, melphalan and cyclophosphamide have been evaluated in ovarian, cervical, colorectal and pancreatic carcinomas. The interferons have some toxicity problems; dose-related side-effects include influenza-like symptoms, lethargy and depression. Myelosuppression, affecting granulocytes, may occur along with cardiovascular problems such as hypotension or hypertension and arrhythmias.

In 1980, researchers at Upjohn showed that a new group of 6-phenylpyrimidine derivatives caused the body to produce interferon. It has now been demonstrated that the same drugs protect animals against viruses and also improve their defences against tumour cells, although this has not yet been demonstrated in humans. It has also been suggested that interferon stimulates prostaglandin synthesis, and this may help to explain how interferon inhibits cell growth.

9.14.2 Tumour necrosis factor

Tumour Necrosis Factor (TNF) is a glycoprotein produced by macrophages, monocytes and natural killer cells, and is partly responsible for tumour cell lysis.

Phase I and II studies, administering the agent either intravenously or intramuscularly, have been carried out on TNF with mixed results. Part of the problem is ensuring that a high enough concentration of TNF reaches the tumour site, but this has to be balanced against adverse effects which include hypotension and cardiotoxicity. Transient fever has also been observed in some patients, as have haematological disturbances, the latter being reversible on cessation of treatment.

9.14.3 Interleukin (Aldesleukin®, Proleukin®)

Interleukin-1 has been shown to possess both direct and indirect antitumour effects. Its main use in the treatment of cancer is for the protection of bone marrow cells from the deleterious effects of radiation and chemotherapy. However, it can also release a cascade of haemopoietic growth factors.

Interleukins are proteins that occur naturally in the body; there are several classes which are being studied for their role in inflammatory and immunomodulatory processes. A recombinant interleukin, interleukin-2 (IL-2), is used clinically in metastatic renal cell carcinoma, where it is administered by intravenous infusion. Unfortunately, the response rate is less than 50% and there is a comprehensive toxicity profile, with one of the most common adverse effects being capillary leakage which leads to hypotension and pulmonary oedema. Investigations are also underway to determine whether IL-2 can enhance the efficacy of tumour vaccines.

9.14.4 Growth factors

Growth factors or cytokines are proteins which affect cell growth and maturation. Recombinant technology has allowed the production of large amounts of cytokines and there are several in clinical trials. Haemopoietic growth factors have found a use in counteracting the myelosuppressive side effects associated with many anticancer agents. Granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) increase the circulating number of neutrophils, eosinophils and macrophages by inducing inflammation, and it has been shown that some tumour cells possess receptors for these CSFs. The dosing regimen is usually once daily, with side effects including influenza-like symptoms. Erythropoietin has also been shown to be clinically useful in treating certain types of malignant anaemia, and is naturally produced by the body in response to hypoxia. Recombinant technology has now been applied to the production of erythropoietin.

Inhibiting growth factors can lead to useful antitumour activity, and known inhibitors include octreotide (Sandostatin®) which is in clinical use, and suramin (a polysulphonated naphthylurea). These two compounds are analogues of somatostatin, a naturally-occurring growth hormone. Octreotide is administered subcutaneously and is useful in controlling symptoms, but does not always cause tumour reduction. Suramin binds proteins extensively due to its polyanionic nature. Early clinical trials with suramin were hampered by severe toxicities (renal and liver dysfuntion, adrenal insufficiency and peripheral neuropathy), but with suitable dosage adjustments clinical trials are continuing in breast and prostate cancer. The clinical use of lymphokine (a protein or glycoprotein produced by a lymphocyte) and natural killer cells is also under investigation.

9.15 PRODRUGS AND DRUG TARGETING APPROACHES

A number of different strategies are available for targeting cytotoxic agents to tumour sites or for activating them inside or near a tumour. Some examples of these are described below. Also see the section on Gene Targeting in 9.17.1.

Mitomycin C (9.60) is a naturally-occurring antitumour antibiotic, considered to be the prototype bioreductive alkylating agent. The three components of the mitomycin molecule essential for its mode of action are the quinone, aziridine and carbamate moieties.

It is thought that initial reduction of the quinone (one-electron reduction yields a semiquinone, whilst a two-electron reduction gives the hydroquinone) leads to transformation of the heterocyclic nitrogen from an amido to an amino form which facilitates elimination of the P-methoxide ion. Tautomerisation of the resulting iminium ion and loss of the carbamate group then creates an electrophilic centre which is susceptible to attack by a nucleophilic DNA base. Nucleophilic attack of the aziridine moiety by a nucleophile on the opposite strand of DNA also occurs, leading to an interstrand cross-link. It is now

(9,60); Bioreduci™ of Mitomycin C (Mitomycin C Kyowa®) follows*) by DNA Cross-lin king known that the main mode of DNA interaction for mitomycin involves cross-linking two guanine-N2 groups within the minor groove. However, the most important feature of mitomycin is the bioreductive "trigger" that is required before cross-linking can take place. It is known that the centres of some tumours, particularly older ones of larger size, are hypoxic due to a poor blood supply. The bioreductive conditions that exist at the centre of these tumours is thought to explain the tumour cell selectivity of mitomycin, which is successfully used to treat solid tumours such as those of the colon, lung and breast. The concept of bioreductive activation has caused great interest, and a number of other compounds have been designed based on this mechanism of action (see Chapter 7).

9.15.1 Bioreductive prodrugs

2. Reaction NH with DMA

2. Reaction NH with DMA

9.15.2 Estramustine

Estramustine (9.61) is a nitrogen mustard that exerts its action by alkylating DNA. The rationale behind this prodrug is that by linking the mustard to oestradiol, the hormone component of the molecule may preferentially transport the drug to those cells which bear oestrogen receptors. Once at the site, the mustard-hormone conjugate may hydrolyse so that the mustard fragment is taken into the cell.

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