New cytotoxic drugs and targets in oncology J.H. Beijnen Introduction

the purpose of increasing the therapeutic efficacy During the past 30 y e a r s c h e m o t h e r a p y has un- of existing drugs. Some valuable analogues h a v e doubtedly exerted a pivotal influence on the de- been introduced, but these cannot be described as velopment of cancer therapy. The i m p r o v e m e n t s real breakthroughs. It should also be noted t h a t in survival r a t e s in the past 2 decades can be at- one of the most effective anticancer drugs, cisplatributed, at least partly, to the clinical use of tin, was discovered by chance. Thus, it is not surchemical agents. In some cases of even advanced prising t h a t there is considerable debate about and disseminated choriocarcinoma, lymphoma, the approaches pursued thus far in the discovery leukaemia, testicular cancer and several child- and development of anticancer drugs, and partihood malignancies, c h e m o t h e r a p y can still offer cularly screening methods [2]. the patient the prospect of cure. Breast cancer, Traditional approaches to new drug developosteosarcoma, soft tissue sarcoma and colorectal m e n t have led to the discovery of a l k y l a t i n g cancer can be treated with a curative intent in agents and other compounds t h a t interfere with the adjuvant setting after radical resection of lo- the functioning of DNA. These drugs act by damcal disease. aging DNA or, in the case of the antimetabolites However, the majority of patients with cancer t h a t inhibit purine or pyrimidine synthesis, by present with clinically evident m e t a s t a s e s or ex- blocking nucleic acid metabolism. It is now clear perience widespread recurrences after local t h a t other, mechanism-based approaches defined t r e a t m e n t (surgery or radiotherapy). In these ca- by t u m o u r biochemistry m u s t be followed as it is ses, local t r e a t m e n t m a y no longer represent a unlikely t h a t the classic empirical strategies will reasonable alternative, and systemic t r e a t m e n t lead to further d r a m a t i c i m p r o v e m e n t s in cancer with chemical agents m a y be indicated. Unfortu- t h e r a p y in the n e a r future. nately, however, such malignancies as gastric New insights into t u m o u r biology and biochemcancer, colorectal cancer, non-small cell lung istry h a v e provided clues for a t t a c k i n g the cancancer, m e l a n o m a and pancreatic cancer are cer cell at the molecular origin of m a l i g n a n t poorly responsive to the currently available anti- transformation. In this concept, DNA r e m a i n s an neoplastic drugs. F u r t h e r advances in cancer attractive target. Interest has shifted, however, t r e a t m e n t will therefore strongly depend upon from DNA as a whole to particular nucleic acid the discovery and/or design of new agents with regions t h a t encode for proteins t h a t play an esactivity against these common tumours. sential role in the regulation of m a l i g n a n t cell After the discovery of nitrogen m u s t a r d as the growth. A case in point is the recent identificafirst chemical agent found to produce responses tion of potent new a n t i t u m o u r compounds t h a t in patients with lymphomas, large drug screen- have a high affinity for minor groove adenine/ ing p r o g r a m m e s were initiated in the late 1950s thymine-rich base sequences within DNA with and early 1960s. Since then, about 20,000 new no intercalation. E x a m p l e s of these compounds chemical entities or m i x t u r e s (usually extracts are the antiviral antibiotic distamycin A, which from n a t u r a l sources) have been screened each originates from Streptomyces distallicus [3], and y e a r for in vitro a n t i t u m o u r activity [1]. Thus, in the synthetic cyclopropapyrolloindole derivathe past 30 years, 600,000 compounds have tives, U-80,244 and U-73,975, analogues of the passed these test panels. Although these extremely potent cytotoxic antibiotic CC-1065 enormous screening efforts have yielded a series (Figure 1) [4]. U-80,244 is undergoing preclinical of therapeutically beneficial drugs, the overall toxicology studies [5] and is now being considered outcome has been disappointing. At present, only for further clinical development. It has been spe43 anticancer drugs are licenced in the Nether- culated t h a t these compounds prevent the DNA lands. Moreover, the r a t e of progress has slowed binding of specific nuclear r e g u l a t o r y proteins, during the past decade w h e n attention was shif- thus inhibiting the transcription of specific genes ted toward the development of analogues, with t h a t are i m p o r t a n t for neoplastic cell growth.

Keywords Bryostatin 1 New cytotoxic drugs Signal transduction Taxol Topoisomerase inhibitors Topotecan Tubulin poisons

J.H. Beijnen: Slotervaart Hospital/Netherlands Cancer Institute, Louwesweg 6, 1066 EC Amsterdam, the Netherlands.

258

Beijnen JH. New cytotoxic drugs and targets in oncology. Pharm Weekbl [Sci] 1992;14(4A): 258-67. Abstract

New agents in the preclinical and early clinical pipeline (phases I and II) are described and some of the problems associated with their development are reviewed. The article focuses on tubu]in poisons such as taxol, topoisomerase inhibitors, such as topotecan, and drugs such as bryostatin 1 and mi]tefosin, which interfere with specific signal transduction pathways involved in malignant cell growth. Accepted June 1992.

P h a r m a c e u t i s c h Weekblad Scientific edition

14(4A) 1992

~

N

H~

OCH3 0

HaC

~t~

N

II

0 Cl

III

1

H

H

Figure 1 Chemical structures of the cyclopropapyrolloindole derivative CC-1065 (I) and its synthetic analogs U73,975 (II) and U-80,244 (III)

This concept would be a unique new mode of action for an anticancer agent [6]. It is interesting t h a t so m a n y novel anticancer compounds originate from natural sources [7]. Examples include taxol, distamycin A, dolastatins [8], bryostatins [9], girodazole [10], and didemnin B [11 12]. Some of these products are extremely potent; dolastatin 10 is effective in vivo at levels of about 1 to 10/Lg/kg. The discovery of these potentially important anticancer drugs, such as bryostatin 1, which interferes with intracellular key enzymes involved in t u m o u r signal transduction pathways, has also opened up avenues for exploring these targets in cancer therapy. Much progress is being made in the exciting field of antisense (DNA/RNA) oligodeoxynucleotides, which block the expression of specific genes [13 14]. It is believed t h a t antisense RNAs bind to oncogene messenger RNAs and thereby inhibit their transcription. In doing so, they knock out the activity of genes t h a t encode proteins t h a t play an essential role in malignant transformation. As a result, replication of the cancer cell is arrested. Recently, Szczylik and co-

O II

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')---C--NH-CH-CH-C--O .... ( ~"

Figure 2 Chemical structure of taxol

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~

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Pharmaceutisch Weekblad Scientific edition

3

workers reported on the preparation of a short, single-stranded antisense DNA t h a t specifically recognizes the oncogene on the Philadelphia chromosome t h a t is involved in the maintenance of the leukaemic phenotype. This antisense DNA stops the replication of these cancer cells in vitro [13]. These experiments have demonstrated the feasibility of gene-targeted a n t i t u m o u r therapy. It is clear t h a t the eventual therapeutic application of these molecules will spark a revolution in the t r e a t m e n t of m a n y gene-related diseases, including cancer. Another exciting, albeit speculative, concept is gene transfer. The first clinical study on the safety and efficiency of retrovirus-mediated gene transfer into h u m a n s has been performed by Rosenberg and co-workers, who initiated the therapeutic application of gene transfer in the field of cancer therapy [15]. Gene therapy and antisense therapy will certainly receive much attention in the near future but will also raise a n u m b e r of problems, including some interesting pharmaceutical challenges. The therapeutic application of these macromolecular compounds will be hampered by rapid in vivo degradation and the difficulty of delivering the compound to the intracellular target at the right time. Formulation and targeting will therefore become an important aspect of the clinical development of these new approaches. This article enumerates some interesting new developments in anticancer drug development. It is not intended as an exhaustive survey, and does not discuss, for example, new antimetabolites, bioreductive alkylating agents, biological response modifiers, growth factors, biochemical modulators, chemoprotectors and radiosensitizers [16-18]. Here, tubulins, topoisomerases and signal transduction pathways have been selected as targets for new cytotoxic drugs. Some of these developments are already in the clinic or on the eve of introduction into the clinic.

Tubulin p o i s o n s Interest in research on tubulin poisons was revived with the recognition t h a t taxol, a tubulin binder, has profound a n t i t u m o u r activity. Taxol (NSC 125973) (Figure 2) is a natural substance derived from the bark of the Pacific Yew tree, T a x u s brevifolia. This tall, slow-growing tree is native to mountain gorges in mixed, evergreen forests lying below 7,000 ft in the Pacific Northwest. The compound was initially isolated and characterized in 1971 by Wani and co-workers and appeared to be the first compound with the taxane ring to have potent a n t i t u m o u r activity [19]. Taxo] has also been isolated from other Taxus species including T a x u s baccata, a European variety which is cultivated as ornamental shrubs [20]. The compound has a unique and totally new mechanism of a n t i t u m o u r action. It enhances both the rate and yield of microtubu]e assembly and stabilizes tubulin polymers against depolymerization. Microtubules are associated with m a n y crucial cellular processes, such as cellular chromosome movement, maintenance of cell form, cellular motility and intracellular trans259

port. Taxol-induced inhibition of the microtu- vestigated and efforts are ongoing to domesticate bules arrests cellular replication at the GJM the trees and cultivate high-yielding plants. Forphase of the cycle [21-23]. This is in contrast with tunately, it appears that the needles also contain the classical antimitotic agents, such as podo- taxol and taxol precursors. Derivation of the phyllotoxin and the Vinca alkaloids, which in- drug directly from these sources or, after a synhibit microtubule polymerization. thetic step, from precursor taxan derivatives is Preclinical studies have demonstrated a broad now being investigated. However, as long as spectrum of activity against B16 melanoma, and taxol s~zitable for h u m a n use is obtained only colon and lung xenografts. Clinical studies began from the bark of the Pacific yew, supplies will be in 1983 and to date, 10 phase I clinical trials a real problem. It has been estimated that 36,000 have been completed. From these studies the trees are necessary to extract enough taxol to toxicity profile of the drug has been documented. t r e a t the 20,000 American patients stricken with Dose-limiting toxicities of taxol include myelo- ovarian cancer each year [33]. The number of suppression, peripheral neuropathy, mucositis trees becomes much larger, of course, if taxol and cardiac disturbances [24 25]. Total alopecia proves to be active against breast and lung canis common and hypersensitivity reactions have cer and worldwide demand grows. In addition to its clinical importance, taxol is also been reported. Considerable enthusiasm with taxol was gene- also a powerful tool for elucidating the mechrated by phase II trials showing impressive objec- anisms of regulation and function of cellular mitive response rates of 21% to 37%. These included crotubules, which appear to be an interesting several complete long-term responses in patients target for an anticancer drug. It can be concluded t h a t taxol is an agent with with ovarian cancer refractory to cisplatin therapy [26-28]. Additional phase II trials are on- major potential in the t r e a t m e n t of ovarian carcigoing in other t u m o u r types such as melanoma, noma. The drug has been described as the most important anticancer drug discovered in the past and renal, breast and lung cancer [29]. Taxol has raised some difficult pharmaceutical decade. Some reservations are, however, in orproblems. The compound is poorly soluble in wa- der. The drug offers no cure and in most cases ter and is currently formulated in a vehicle of has produced only partial responses. Never50% polyoxyethylated castor oil (Cremophor EL) theless, the drug offers a potentially beneficial and 50% ethanol. Prior to administration this so- therapy for patients with platinum-refractory lution is diluted in 0.9% sodium chloride. Hyper- ovarian cancer for whom no further therapeutic sensitivity reactions are encountered in 5% to alternatives exist. Still to be defined are the opti10% of treated patients. It is still unclear mal dose and infusion duration of taxol, and the whether these reactions are caused by taxol itself drug's therapeutic efficacy as first-line treator the excipients of the formulation. Prolonging ment and in combination regimens with optimal the drug infusion appears to reduce but not obvi- scheduling [34]. ate the risk of hypersensitivity reactions [30]. P r e t r e a t m e n t with dexamethasone, diphenhy- Topoisomerases The double helix structure of DNA hampers dramine and cimetidine or ranitidine is necessary to decrease the incidence of these reactions. such processes as ~-eplication, recombination and For infusion, polyvinyl chloride-free bags and transcription which are vital for survival of the lines must be used because of the leaching of cell. Within the living cell these topological probplasticizers from these materials by the surfac- lems are solved by the action of enzymes, the tot a n t Cremophor EL. In-line filtration is manda- poisomerases. DNA topoisomerases are ubiquitory because of the danger of microcrystalli- tous intranuclear enzymes which transiently zation after dilution of the formulation in an break and rejoin DNA strands and thereby moaqueous solvent. Therefore, it is recommended dify and unravel the intertwined strands of DNA t h a t taxol solutions be stored in a glass or poly- [35-37]. Two types of topoisomerases (I and II) olefin container and delivered through a poly- have been characterized. The most important difethylene-lined intravenous administration set ferences between these types are listed in Table and in-line filter [31]. The synthesis of more I. Topoisomerase I induces the breakage of one water-soluble new taxol derivatives, in which the strand of DNA and topoisomerase II cleaves a Cremophor EL/ethanol formulation become su- pair of strands of a duplex DNA in concert. Many drugs appear to interfere with the perfluous, is being actively pursued. There is a finite supply of taxol and consider- breakage-reunion reactions catalyzed by DNA able difficulties can be expected in procuring suf- topoisomerases. The enzymes are blocked by ficient amounts of the drug. Supplies are now these agents by stabilization of the 'cleavable limited because m a n y trees must be harvested complexes,' in which the enzyme is covalently and new trees require m a n y years to reach matu- linked to the 8'-(topoisomerase I) or 5'-(topoisority. The development of alternative ways of pro- merase II) termini of single- or double-strand ducing taxol is therefore emerging as the highest DNA breaks. It is not yet exactly clear how the priority. It is clear from the complex chemical topoisomerase inhibitor binds to the complex and structure of the compound (Figure 2) t h a t total how cell killing is established, but the production chemical synthesis will be extremely difficult to of a drug-stabilized enzyme-DNA complex is achieve. The search for semi-synthetic analogues probably essential. Continuous exposure also may be a fruitful approach, with taxotere repre- seems to be important as the drug complexes are senting the first clinically active derivative [32]. reversible and after drug removal cells m a y conProduction of taxol in cell cultures is being in- tinue to divide. Topoisomerases are cell cycle260

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Table 1 Differences between topoisomerases I and H

Structure DNA strand scission DNA strand passage Covalent linkage to Need ATP for activity Implicated in

Topo I

Topo II

monomer single single 3'-terminus no gene transcription

homodimer single and double double 5'-terminus yes replication transcription recombination repair

specific cytotoxic drugs. Brief exposure to these agents produces little or no cell killing and quiescent cells are refractory. The recognition that the plant alkaloid camptothecin (Figure 3) has strong antitumour activity and that the drug exerts its effect exclusively by inhibition of topoisomerase I has aroused interest in this target. It has been proposed that camptothecin inhibits topoisomerase I by interfering with the rejoining reaction of this enzyme [35]. Unfortunately, clinical evaluation of camptothecin had to be discontinued because of the drug's toxicity profile, which includes myelosuppression, gastrointestinal toxicity and haemorrhagic cystitis [38]. CPT-11, a more water-soluble analogue of camptothecin is currently being investiFigure 3

N

Chemical structure o f camptothecin

O O bH'b

gated clinically and its schedule dependency is being studied [39]. Another topoisomerase I inhibitor, currently in phase I clinical trials in the Netherlands, is topotecan. The structure of this novel drug is depicted in Figure 4. Topotecan is not stable at physiological pH and equilibrium processes favour the

CH3 I .,/N'~cH3 HO~_~,~~

~ H3 O

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0

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OH-

Figure 4

H3C~t "OH\~Q A

Chemical structures and equilibrium reactions between topotecan (A; S K & F 104864-A) and its hydrolysed form (B; S K & F 105992)

14(4A) 1992

Pharmaceutisch Weekblad Scientific edition

hydrolysis of the lactone ring to yield the hydroxy-acid SK&F 105992 (Figure 4) [40 41]. Only the closed lactone form inhibits topoisomerase I. Phase II trials with topotecan in patients with small cell lung cancer and patients with colorecta] cancer will be starting soon. Treatment of patients with colon cancer with a topoisomerase I inhibitor looks attractive as it has been demonstrated that topoisomerase I levels are significantly higher in human colon cancer cells than in normal cells [42]. Although topoisomerase I inhibitors have demonstrated antitumour activity in patients it still remains unclear whether topoisomerase I is the sole target for this class of compounds, as DNA topoisomerase I appears to be a nonessential protein. This is in contrast with topoisomerase II, which is indispensable for survival of eukaryotic cells and can be regarded as a nuclear marker for cell proliferation. Only a few years ago it was recognized that many cytotoxic drugs, including doxorubicin, etoposide, teniposide, mitoxantrone, actinomycin D, and m-amsacrine interact with DNA topoisomerases. All of these drugs affect topoisomerase II by stabilizing an abortive topoisomerase-DNA cleavable complex. In these cases, however, it is also not yet clear how the cell killing is associated with the drug-cleavable complex interaction. The recent recognition of topoisomerase II as an important target has provided new perspectives on the pharmacology of these compounds, including the importance of scheduling. High response rates have been reported in the treatment of small cell lung cancer after prolonged daily oral administration of etoposide with maintenance of low serum concentrations [43]. However, assessment of the optimal schedule of administration appears difficult; for example, there is still debate about the optimal mode of administration of doxorubicin [44 45], which has for many years been one of the most widely used anticancer drugs. Since many agents with diverse activity in many different tumour types all share a common intracellular target in the form of DNA topoisomerases, this is an interesting concept for further research. A number of points require additional study, including the schedule dependency of this class of compounds. Combinations of topoisomerase I and II inhibitors may also be an interesting approach to completely eliminating topoisomerase activity in the dividing cell. The results of phase II studies must be awaited before any conclusion can be drawn about the clinical importance of topoisomerase I inhibitors.

Nignal transduction Differentiation and proliferation of normal cells are processes regulated by a complex network of growth-encouraging and growthinhibiting intracellular signalling pathways. The proteins (growth factors, receptors, enzymes, regulatory proteins) involved in these processes are encoded by growth-promoting proto-oncogenes and growth-constraining tumour suppressor genes working as a team in balance. The suppressor genes normally restrain tumour development [46]. Uncontrolled malignant growth arizes 261

when the signalling pathways are deregulated and subverted by genetic aberrations of the proto-oncogenes that encode for the proteins involved in these pathways. Proto-oncogenes, the progenitors of oncogenes, are normal genes that can be altered or overexpressed by genetic changes and thereby transformed into oncogenes. The proteins encoded by the oncogenes have lost important regulatory constraints, leading to altered signal transduction and tumourigenesis. During the past few years, it has become increasingly clear t h a t peptide growth factors, their receptors, and other intracellular regulatory proteins play a crucial role in the control of cell differentiation, proliferation and malignant transformation. Studies on the fundamentals of how external stimuli are transmitted from the cellular membrane to the nucleus are therefore extremely important as they may lead to the development of new modalities for the treatment of cancer [47]. Growth factors are very active biologicals and can interact specifically with membrane-bound receptors. The binding of growth- and differentiating-controlling ligands to specific receptors at the cell surface sets in motion a complex cascade of changes in phospholid metabolism, ion flux and protein phosphorylation. These changes trigger intracellular signals resulting in differentiation and proliferation [48]. Although there are multiple intracellular mitogenic pathways, those with growth factor receptors with tyrosine kinase activity and receptors with serine/threonine activity kinase activity have been studied most extensively [49]. Protein kinase C p a t h w a y

Neuropeptide mitogens such as bombesin, angiotensin, vasopressin, bradykinin and vasoactive intestinal peptide (VIP) bind to transmembrahe receptors, which results in an increase in serine/threonine kinase activity (Figure 5). Following ligand-receptor binding, the mitogenic signal is transduced by a membrane-bound guanine nucleotide binding protein (G protein) which exchanges guanosine diphosphate for gua-

~

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Figure 5 Simplified scheme illustrating the main steps in the protein kinase C signalling pathway. GF: growth factor; R: receptor; G.Prot: G-protein; GTP: guanosine triphosphate; GDP: guanosine diphosphate; PLC: phospholipase C; PI(4,5)P2: phosphatidylinositol 4,5-biphosphate; I(1,4,5)P3: inositol 1,4,5-triphosphate; DG: diacylglycerol; PKC: protein kinase C; prot.: protein; prot.P: phosphorylated protein

262

nosine triphosphate and thereby obtains its active configuration. This activation results in the activation of the G protein coupled effector phosphoinositide phospholipase C (PLC). This protein, in turn, catalyses the hydrolysis of a specific membrane-bound phospholipid, phosphatidylinositol 4,5-biphosphate (PI(4,5)P2), to generate inositol 1,4,5-triphosphate (I(1,4,5)P 3) and 1,2-diacylglycerol (DG). The resulting products are thought to act as endogenous second messengers, where DG is a stereospecific direct activator of protein kinase C (PKC). It is believed this activation occurs with simultaneous translocation of the enzyme from the cytosol to the plasma membrane. Here it is involved in the phosphorylation and activation of membrane bound proteins leading to a certain cellular response (Figure 5). PKC is a large family of phospholipid-Ca2+dependent serine-threonine kinases involved in multiple signalling pathways by phosphorylation of a range of intracellular seryl and threonyl residues of protein targets that cause alterations of the cellular programme of gene expression important for the mitogenic response. Seven subspecies of PKC have been identified thus far [50] and diacylglycerols are the main physiological activators. I(1,4,5)P 3 acts by releasing Ca 2+ sources and thereby activates calcium-dependent protein kinases such as the PKC family. A corollary of the hypothesis that unregulated expression of the components in the signalling pathways is implicated in malignancy is that intervention in these pathways may be an effective approach in cancer therapy. Theoretically there are several stages at which therapeutic intervention might be successful. Firstly, the signal transduction process can be interrupted at various prereceptor levels. The first step in the PKC signalling pathway is the interaction of the growth factor with its receptor (Figure 5). Neutralization of the growth factor would stop the growth stimulus. This concept has been utilized in bombesin-producing lung cancers. Bombesin is a small tetradeca-neuropeptide growth factor secreted by human small cell lung cancer (SCLC) cells. Neuroectodermally-derived SCLC cells can produce and secrete their own regulatory growth factors (autocrine growth factors), such as bombesins and gastrin-releasing peptide (GRP). Blockade of the autocrine action of this tumor cell mitogen by antibodies raised against bombesin inhibits the in vitro clonal cell proliferation of SCLC cells, as well as the growth of xenografts in nude mice [53]. An alternative approach to intervention in this signalling pathway is to disrupt the growth factor-induced growth with a receptor anta'gonist. Such antagonists bind to the receptor but do not activate the signal transduction. Bombesinmediated mitogenesis has been blocked by different antagonists with high specificity [54-56]. Figure 6 shows the amino acid sequences of bombesin and a peptide antagonist, which effectively inhibits the mitogenesis induced by GRP and other bombesin-like growth factors. A limitation of these approaches is that antibodies and peptide-antagonists are rapidly degraded by pep-

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14(4A)1992

pGlu

G l n A r g L e u G l y A s n G l n T r p A l a V a l G l y H i s L e u M e t NH 2

D-Arg

P r o Lys Pro D - P h e

Figur e 6 A m i n o acid sequence of bombesin (I) a n d a b o m b e s i n antag o n i s t (II) [55]

Figure 8 C h e m i c a l structure o f staurosporine H

"OCH3 H

CH3

Figtlre 9 C h e m i c a l structure o f bryostatin 1

Gln D-Trp

Phe D - T r p

L e u L e u NH 2

II

tidases in vivo. Furthermore, other pathways t h a t are probably involved in growth are not completely inhibited by antagonists, indicating t h a t multiple blocking activity is necessary to achieve a total arrest of cell growth. PI(4,5)P 2 is believed to play an important role in mitogen-induced signalling (Figure 5). Matuoka and co-workers developed and microinjected an antibody to PI(4,5)P 2 into the cytoplasm of NIH 3T3 cells and found t h a t it abolished cell proliferation induced by bombesin and platelet-derived growth factor [57]. Whether this highly experimental in vitro technique can be translated to the clinic in the future is very doubtful. A class of promising new anticancer drugs are the ether lipids, which are analogues of platelet activating factor. ET-18-OCH 3 is the prototype of this group of agents, and related compounds (BM 41.440, SRI 62-834, hexadecyl-phosphocholine, ara-CDP-DL-PTBA; Figure 7) have been synthesized [58]. The ether lipids express their a n t i t u m o u r activity by multiple effects at the cell m e m b r a n e level. They interfere with the binding of growth factors to their receptors, disturb lipid metabolism, affect calcium channels and increase m e m b r a n e fluidity. The exact molecular mechanisms are, however, poorly understood at the moment, but it is clear t h a t the effects involve solely the cell membrane and neither DNA nor DNA synthesis [52 58]. E t h e r lipids depress membrane-bound PLC activity. This leads to a decrease in the formation of I(1,4,5)P 3 and, consequently, reduced calcium release from intracellular stores (Figure 5). This effect is probably the cause of inhibition of PKC activity by the ether lipids. Several ether lipids are now in phase I and II clinical trials [58 59]. A solution of hexadecylphosphocholine (miltefosine) in a mixture of alkylglycerols has been successfully used for the topical t r e a t m e n t of skin metastases or local recurrences from breast cancer [60]. O

CH30"~~

H7C3

OH3 CH3

O

Because PKC plays a pivotal role in the intracellular regulatory transduction pathways of growth-promoting stimuli, it is an attractive target in the search for novel antineoplastic agents [51]. This interest arose after the recognition of the regulatory domain of PKC as the main target for phorbol esters. These natural products, derived from Croton tigium, exert a high activity as tumor promotors by mimicking the action of the putative natural activator DG. Furthermore, it has been demonstrated t h a t overproduction and dysregulation of PKC results in disordered and uncontrolled growth of cells in vitro [61 62]. PKC antagonists may thus be useful as therapeutic anticancer agents, which justifies the efforts put into the development of agents which interfere with PKC activity. The microbial alkaloid staurosporine is a very potent and selective inhibitor of PKC (Figure 8). One of the most intriguing classes of agents with PKC activity are the bryostatins [63]. Bryostatin 1 (Figure 9) is a macrocyclic lactone isolated from the marine bryozoan Bugula neritina; a series of similar compounds have been isolated with different ester side chains. Bryostatin 1 is very potent in murine t u m o u r models and possesses an interesting pharmacological profile as it appears to be a nontumour-promoting activator of PKC when it binds to the regulatory domain of the enzyme. This observation may sound paradoxical in view of the action of phorbol esters as t u m o u r promoting-activators of PKC. However, bryostatin 1 induces downregulation of PKC. Furthermore, the differences between PKC activators may be explained by the existence of individual subspecies of PKC, which may have different functions and differential distribution in various cells and even within the same cell. Various drugs may also have differential inhibitory or stimulatory effects on the various subspecies of PKC. More research is w a r r a n t e d to gain insight into this phenomenon [64]. A clinical trial of bryostatin 1 is now underway in the UK. The drug has been isolated only in milligram quantities, but because of its high activity, this amount is probably enough to complete a phase I trial. The starting dose in the phase I clinical trial is 5 ~g/m 2 [63]. Interestingly, tamoxifen is also a modulator (inhibitor) of PKC and this may explain the efficacy of the compound in oestrogen-receptor negative m a m m a r y tumours. PKC is ubiquitous in m a m m a l i a n tissues and it will be very difficult to design compounds t h a t act selectively on tumours cells. However, increasing understanding of the biology of PKC and its isoenzymes m a y lead to strategies t h a t offer t u m o u r specificity [50]. Knowledge of the characteristics of the binding sites of bryostatin 1 and the phorbol esters would be very helpful in this respect.

Tyrosine kinase pathway Ligand binding to tyrosine growth factor receptors leads to the activation of the intracellular tyrosine kinase domain of the receptor t h a t catalyses the transfer of phosphate from ATP to tyrosine residues of peptide substrates (Figure

H

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I

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263

Figure 7

Chemical structures o f ether lipids. I: ET-18OCH3; II: B M 41.440; III: S R I 62-834; IV: hexadecylphosphocholine; V: araCDP-DL-PTBA [58]

H2C O (CH2)17--CH3 I HC--O OH3

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HC--CH 2

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11

O--CH 3

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~ ~ H2OC18H37 H20\/OCH2CH2~N(CH3)3 o~o

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H2C--O--P--O-- ,P--O I

OQ

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OQ

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OH 264

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14(4A) 1992

[

OH

__]

IGF I

i

T OH 0

prot.

Figure 10

Simplified scheme illustrating the tyrosine kinase pathway. GF: growth factor; R: receptor with tyrosine kinase activity; prot.: protein; prot. P: phosphorylated protein

[

membrane

R

prot. @ -

-

--,- Cellular response

10). Various enzymes, including PLC and phosphatidylinositol 3' kinase (PI-3K), can be activated through phosphorylation by the activated receptor tyrosine kinase [47 49]. Since many growth factor receptors involved in cell proliferation and transformation function as protein tyrosine kinases (PTK) to transduct external growth stimuli to the nucleus, considerable research has been devoted to the development of compounds that intervene in these pathways. Platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and insulin-like growth factor can act as ligands for these receptors. Suramin competes with the binding of these growth factors and possesses antitumour activity. This polysulphonated naphthylurea derivative (Figure 11) has been used

NIH CO

NIH CO

T

OH

OH 0

ii

Figure 12 Chemical structures of genistein (I) and quercetin ([I)

}---SO3Na

NaO3S

~J~'OH

S03Na

Studying a series of related flavonoids, Akiyama et al. [70] found that the isoflavone genistein (Figure 12), isolated from P s e u d o m o n a s fermentation broth, possessed the highest PTK inhibiting activity of the EGF receptor. Another flavonoid, quercetin (Figure 12), was found to inhibit the growth of a multidrug-resistant estrogen receptor-negative MCF-7 human breast cancer cell line [71]. Unfortunately, genistein and other PTK inhibitors, such as erbstatin, appeared to be too toxic for further development [72]. The development of new analogues must be awaited before the importance of this group of agents in cancer therapy can be judged. Conclusion

Recent research in tumour biology and molecular biology has enormously extended our knowledge of the mechanisms underlying malignant transformation. This new knowledge has led to the identification of many new potential target sites that could be exploited for anticancer drug development. Many highly potent compounds have already been developed. However, the main objective, the design of cytotoxic agents that are fatal to every tumor cell but have no toxicity for normal cells, appears to be very difficult. A full understanding of the biochemical differences between cancer cells and normal cells is essential for further development of cytotoxic drugs that exploit these differences. Much progress has been made but there also remains much to be done. References

F i g u r e 11 Chemical structure of the hexa-

sodium salt of suramin

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New cytotoxic drugs and targets in oncology.

New agents in the preclinical and early clinical pipeline (phases I and II) are described and some of the problems associated with their development a...
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