Review. Topoisomerase I inhibitors: Review and update. M. L. Rothenberg

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1 Annals of Oncology 8: , O 1997 Kluwer Academic Publishers. Printed in the Netherlands. Review Topoisomerase I inhibitors: Review and update M. L. Rothenberg Division of Medical Oncology, University of Texas Health Science Center at San Antonio, TX, USA Summary This review presents a summary of preclinical and clinical data on the topoisomerase I (topo I) inhibitors that are under clinical development. To date, all of the topo I inhibitors that have been clinically evaluated are analogues of camptothecin, an extract of the Chinese tree Camptotheca acuminata. The therapeutic development of camptothecin was initially limited by its poor solubility and unpredictable toxicity. More recently, a number of water-soluble camptothecin analogues have undergone extensive evaluation and have demonstrated significant clinical activity. These include irinotecan (CPT-11), topotecan, and 9-aminocamptothecin (9-AC). Preliminary data are also reviewed on other camptothecin analogues (GG- 211 and DX-8951f), on oral formulations, and on non-camptothecin topoisomerase I inhibitors. The topoisomerase I inhibitors have already demonstrated a broad spectrum of antitumour activity, most probably due to their unique mechanism of action and lack of clinical cross-resistance with existing antineoplastic compounds. The challenge for the next five years is to identify ways to integrate the topo I inhibitors into multidrug and multimodality therapies to achieve optimal antitumour effect, while keeping the side effects of these therapies manageable. Key words: camptothecin analogues, cancer, CPT-11, irinotecan, topoisomerase I inhibitors, topotecan Introduction Effective cancer therapy evolves from the identification of a molecule or process that can be targeted by a therapeutic agent in a relatively selective and exploitable manner. In the early 1970s, such a discovery was made with the identification of topoisomerase I (topo I). Over the past two decades, there has been an explosion of interest in topo I and the development of compounds that can selectively inhibit this enzyme. This review presents an overview of the development of topo I inhibitors and a summary of the current status and future potential of topo I inhibitors in clinical practice. allows passage of newly synthesized DNA without it becoming irretrievably tangled in the parent strands. Topoisomerase I subserves this process through a reversible trans-esterification reaction which yields a covalent intermediate form with the tyrosine of the enzyme bound to the 3' end of the DNA strand. This 'cleavable complex' normally lasts only long enough to allow passage of the newly synthesized strand through the cut, after which topo I reseals the cleavage (Figure 1). Whereas topo I performs this function on a single DNA strand, a separate enzyme, topoisomerase II, catalyzes the opening and closing of two strands of DNA, either simultaneously or sequentially. Topoisomerase I function The topoisomerases were discovered in 1971 but it was not until the 1980s that the significance of these enzymes as potential therapeutic targets was appreciated [1]. Topoisomerase I plays a crucial role in the normal replication of DNA. In its physiological state in the chromosome, the DNA helix is supercoiled and tightly packed into chromatin. Replication requires transient relaxation and unwinding of the parent DNA to allow the replication fork to proceed down the DNA strand and serve as a template for synthesis of new strands of DNA. In order to achieve this without creating extreme torsional stress on the parent DNA, transient cleavage of the DNA is required. This transient cleavage also Camptothecins: The first specific topoisomerase I inhibitors As part of a large natural product screening effort, an alkaloid extract of the tree Camptotheca acuminata, termed camptothecin, was identified in 1966 as having significant antitumour activity in the L1210 and P388 murine leukaemia models [2]. Although rapidly introduced into clinical trial, it was not until the early 1980s that the mechanism of antitumour activity was identified as inhibition of topo I [3]. In fact, camptothecin was the first specific topo I inhibitor to be discovered. The cytotoxic effect of camptothecin and its analogues can be explained by the fork collision model [4] illustrated in Figure 2. In this model, camptothecin

2 838 Increasing lension and supercoiling of DNA Topoisomeraae 1 binds to one DNA strand and cuu H (cleavage reaction). 9-NC 9-nitrocamptoihetine 9-AC: 9-aminocamptothccin l-methylenedtoxycamplolhecin The intact strand of DNA passes through the nick, resulting in the relaxation of ihe torsiona! strain Topoisomera.se 1 repeals the broke: strand ireligalion step) and dissocii from ihc DNA moletule 7 A'-meihylpiperazinomeihyl-If). I l-etriylenedioxy-2q-5-camp(olhecin GG-21I fgi I l h Figure 1. The mechanism of topoisomerase I action. Figure 3. Chemical structure of camptothecin and its principal analogues; the structure of SN-38 (active metabolite of CPT-l I) is also shown. Development of water-soluble camptothecin analogues Top I = Topoisomera\e I C = Camptoihecin Solid lines = Parenl DNA Dolled lines = Daughter DNA Figure 2. Collision of the replication fork with the camptothecinstabilized cleavable complex results in an irreversible double-strand break in the DNA. Clinical development of intravenously-administered camptothecin sodium began at the National Cancer Institute in the late 1960s. Although some antitumour activity was observed, severe and unpredictable toxicities (principally myelosuppression and haemorrhagic cystitis) prevented further clinical development [7-9]. A search for analogues of camptothecin was begun with the aim of overcoming the two key limiting factors in development of the parent drug: high toxicity and low solubility in water. This resulted in the discovery of a number of water-soluble camptothecin analogues that have now reached the clinic: CPT-l 1 (irinotecan) [10], topotecan [11], 9-aminocamptothecin (9-AC) [12, 13] and GI (GG-211) [14, 15]. Among these, CPT-l 1, topotecan and 9-AC are currently farthest along in clinical development. Preclinical studies binds to and stabilizes the normally transient cleavable DNA-topo I complex that forms during replication. Although the initial cleavage action of topo I is not affected, camptothecin inhibits the religation step that normally reseals the parent strand of DNA after passage of the daughter strand. The collision of the replication fork with this cleaved strand of DNA causes an irreversible double-strand DNA break which arrests the process of cell division and results in cell death. These events occur during the S-phase of the cell cycle and, historically, camptothecins have been considered to be cellcycle specific drugs. Recent data, however, suggest that the camptothecins may possess cytotoxic effects beyond those exerted while cells are in S-phase. Camptothecininduced cytotoxicity has been observed in cells not actively synthesizing DNA [5]. Replication-independent mechanisms of cytotoxicity may involve the induction of serine proteases and endonucleases [6]. The chemical structures of camptothecin, CPT-l 1, topotecan, 9-AC and GG-211 are shown in Figure 3. CPT-l 1 is metabolized by carboxylesterases to an active metabolite, SN-38, the structure of which is also shown in Figure 3. The in vitro and in vivo activities of CPT-l 1, topotecan and 9-AC have been demonstrated in a variety of murine and human tumour xenograft models. Both CPT-11 and topotecan are effective in vivo against L1210 and P388 leukaemia, Lewis lung carcinoma, mouse colon adenocarcinomas 38 and 51, mouse breast adenocarcinoma 16/C, as well as human tumour xenografts B16 melanoma and human HT-29 colon adenocarcinoma [16-23]. CPT-11 also has demonstrated activity against S 180, meth-a fibrosarcoma, Ehrlich carcinoma, MH 134 hepatoma, mammary carcinomas C3h/HeN, pancreatic ductal carcinoma 03, and in human tumour xenografts

3 839 Table 1. Summary of comparative in vitro cytotoxicity and DNA damage caused by the principal camptothecin derivatives [27]. CAM CPT-11 SN-38 TPT 9-AC HT-29 cytotoxicity (IC50[nM]) 10.0 > DNA damage in whole cells (concentration [um] equivalent to 1000 rads) > DNA damage in nuclei (concentration [um] equivalent to 1000 rads) > Abbreviations: CAM - camptothecin; TPT - topotecan; 9-AC - 9- aminocamptothecin; CPT-11 - irinotecan. colon Co-4, mammary MX-1, gastric St-15, St-16 and squamous lung carcinoma QG56 and neuroblastoma and neuroectodermal tumour xenograft models [24]. CPT-11 and topotecan have also been assayed in the Human Tumour Stem Cell Assay. For topotecan, activity was observed against ovarian, renal cell, and breast cancer specimens at relatively high drug concentrations [25]. For irinotecan, activity against colorectal, ovarian, non-small cell lung, breast, mesothelioma, sarcoma, pancreas, and cervical cancers was observed at lower drug concentrations [26]. The in vitro activity of these compounds has been directly compared by Tanizawa et al. [27], and the results of cytotoxicity and DNA damage assays are summarized in Table 1. They show a potency ranking as follows: SN-38 > camptothecin > 9-AC > topotecan > CPT- 11. CPT-11 required metabolic conversion to SN-38 before it showed any significant activity, indicating that CPT-11 serves as a prodrug for SN-38. Whether topo I content is directly correlated with sensitivity to topo I inhibitors is an area of controversy. Higher topo I levels have correlated with sensitivity to topo I inhibitors, while decreased levels have correlated with topo I resistance in some in vitro models [28-30]. Others, however, have found topoisomerase gene copy number and protein expression to be inversely correlated with sensitivity to SN-38 in vitro in several breast and colon tumour cell lines [31]. In human tumour specimens, topo I expression and activity have been found to be higher in colorectal, prostate, and ovarian cancers than in adjacent normal tissue, but no such difference was detected in renal cell cancers [32-35]. It is very likely that gross measurement of topo I content or activity will not be sufficient to fully explain the sensitivity or resistance of a given tumour to camptothecin analogues. Other factors likely to determine the relative potency of camptothecin analogues include 1) intrinsic topo I inhibitory activity, 2) drug half-life, 3) interaction with plasma proteins, 4) ratio of lactone (active) to carboxylate (inactive) forms of the drug in the circulation, 5) relative susceptibility of the drug to efflux from the cell via p-glycoprotein, 6) stability of binding to topo I, 7) ability of the cell to undergo apoptosis, 8) effectiveness of DNA repair mechanisms, and 9) status of cell-cycle regulatory processes [17, 36, 37]. These factors will be dealt with later in this review in the section on Mechanisms of Resistance to Camptothecins. Clinical development of camptothecin analogues CPT-11 and topotecan have undergone the most extensive clinical evaluation. 9-AC has recently been reformulated and entered phase II studies in the US. GG-211, oral camptothecin, and 9-nitrocamptothecin have undergone phase I and, in some cases, limited phase II evaluation. Clinical data concerning each compound are reviewed below. CPT-11 (irinotecan) Pharmacokinetic studies CPT-11 must undergo hydrolysis or de-esterification to form the active metabolite, SN-38. Elimination occurs primarily in the bile and secondarily in the urine. SN-38 glucuronide has recently been identified as an important inactive metabolite of SN-38 [38]. Sixteen additional metabolites have recently been characterized in the plasma and blood and these may provide additional insights into other mechanisms of metabolism for this compound [39, 40]. The terminal half-lives for both CPT-11 and SN-38 are long: 7.9 to 14.2 hours for CPT-11 and 13.0 to 13.8 hours for SN-38 [41, 42]. The pharmacokinetics of CPT-11 are linear with increasing dose, and the total body clearance (14.3 to /h/m 2 ) and the volume of distribution (157 1/m 2 ) are not affected by dose or regimen. The pharmacokinetics of SN-38 are somewhat more complex. While some groups have found a proportional relationship between CPT-11 dose and SN-38 Cpmax and the area under the plasma concentration-time curve (AUC) [43], others have not [42, 44]. The reasons for this variability are not well understood at present, but may be related to the different schedules and doses used. In those studies in which it has been tested, approximately 34% of the total CPT-11 and 45%-64% of the total SN-38 is found in the lactone (active) form [42]. Phase I studies Phase I studies with CPT-11 were initially conducted in Japan and subsequently in the US and France. Schedules of administration tested include 5-14-day continuous infusions [44, 46]; afive-daycontinuous hepatic arterial infusion [47]; a five-day oral dosing schedule [48]; a 30- to 90-minute infusion weekly [42, 49, 50]; a 30- to 90-minute infusion once every three weeks [41, 51, 52] and a daily infusion for three consecutive days every

4 840 Table 2. Phase II studies with CPT-11 in colorectal cancer. Tumour type CPT-11 regimen n entered; evaluable Responses and response rate (evaluable patients) Reference CRC CRC CRC CRC CRC CRC One 5-FU based One 5-FU based One 5-FU based CT CTorRT 350 mg/m 2 q3w 350 mg/m 2 q3w 350 mg/m 2 q3w mg/m 2 qlw* 125 mg/m 2 qlw 125 mg/m 2 qlw 125 mg/m 2 qlw 150 mg/m 2 q2w 165; 130 b 48; 48" 107/95" 48,43 41; 21 25; 13 41; 41 0/, Oj 23/130(17.7%) 9/48(18.8%) 13/95(13.7%) 10/43 (23.2%) 5/21 (24.0%) 2/13(15.0%) 13/41 (31.7%) 13/52(25.0%) 4/11(36.4%) [75] [76] [44] [77] [78] [74] The US weekly regimens were administered four weeks on, two weeks off; the Japanese weekly schedule is without a rest period. Abbreviations: CT - chemotherapy; RT - radiotherapy; qlw - weekly; q2w - every two weeks, etc.; NIC - not known; CRC - colorectal cancer. * Treatment started at 150 mg/m 2 qlw and reduced to 125 mg/m 2 qlw after four of the first eight patients had grade 4 diarrhoea. " Eligible patients. three weeks [53]. The dose-limiting toxicity (DLT) with all schedules is delayed diarrhoea and/or neutropenia. Different schedules have been recommended for phase II studies in different countries. In Japan the recommended schedule is 100 mg/m 2 /week with breaks taken for grade 2 or greater toxicity; in the US the recommended schedule is 125 mg/m 2 /week for four weeks followed by two weeks rest, and in Europe the recommended schedule is 350 mg/m 2 administered once every three weeks. During phase I studies, responses were observed in patients with colorectal cancer, non-small-cell lung cancer (), mesothelioma, breast cancer, oesophageal cancer, renal cell cancer, cervical cancer, ovarian cancer, lymphoma and leukaemia [54]. A number of phase I studies have been performed combining CPT-11 with CDDP using a variety of different schedules [55, 56]. The DLT of this combination appears to be myelosuppression, diarrhoea or both, regardless of the schedule used. In one study, the use of granulocyte colony-stimulating factor (G-CSF) permitted a higher dose of CPT-11 to be administered [57]. Peak plasma concentration of SN-38 correlated well with both myelosuppression [58] and diarrhoea [59]. Phase II studies of the combination are considered later in this review. A combination of CPT-11, CDDP and vindesine was associated with a similar toxicity profile to that of the combination of CPT-11 and CDDP, but reportedly also with severe liver toxicity [60]. When given in combination with CPT-11, VP-16 has a higher maximum concentration and AUC than when given alone [61], suggesting the possibility of a drugdrug interaction. This combination also appears to have substantial activity against, although toxicity, especially neutropenia, is pronounced. Administration of G-CSF appears to be important in the maintenance of dose-intensity and clinical activity for this drug combination [62-65]. The interaction between CPT-11, 5-FU and leucovorin appears to be complicated [66-68]. Preclinical studies have demonstrated various results, from subadditivity to synergy, with drug sequence having a significant impact on this interaction [69]. The addition of leucovorin to the CPT FU combination appears to alter this relationship and enhance the cytotoxicity of the combination [69]. Simultaneous administration of CPT-11 and 5-FU has been reported to result in a reduced AUC of SN-38 suggesting that 5-FU or a metabolite may interfere with the metabolic conversion of CPT-11 to its active form [67, 70-73]. If this is indeed the case it may be necessary to separate their administration during a combination protocol. Colorectal cancer Phase IIclinical trials Phase II data have been accumulated for CPT-11 in the treatment of a wide variety of tumour types, but the most extensively studied is single-agent therapy of advanced colorectal cancer (Table 2). The first report of activity in patients with colorectal cancer was published by Shimada et al. in Japan [74]. In a somewhat heterogeneous group of patients, they observed 17 partial responses in 63 patients (RR = 27%, 95% CI = 16%- 38%). In the subset of patients who had received prior chemotherapy with 5-FU, there were 10/46 responses (RR = 22%). Using a four weeks on, two weeks off schedule, Rothenberg et al. in the United States reported 10 responses (one CR, nine PR) in 48 patients with colorectal cancer that had progressed during or within six months of one prior 5-FU based regimen (RR = 21%) [44]. In the largest of the studies reported to date, Rougier et al. in France treated 213 patients using the 350 mg/m 2 once every three weeks schedule [75]. An objective response rate of 17.7% was observed in the subset of patients who had received one prior 5-FU-based regimen. The response rate did not differ for those patients who had progressed while receiving 5-FU-based chemotherapy compared to those who had disease progression after having received similar therapy. Of note is that 34% of all patients with progressive disease at study entry were free of disease progression

5 841 at six months. A subsequent study using the same schedule in centers throughout Europe reported similar results [76]. A similar consistency across studies has been observed in patients with metastatic colorectal cancer treated with CPT-11 as first-line therapy. Response rates of 19%-36%have been reported in this group of patients with a median survival of months [75, 77, 78]. These results are quite similar to those that can be expected with bolus 5-FU + leucovorin. Taken together, these studies provide important evidence that CPT-11 has significant clinical activity in patients with advanced colorectal cancer, including those whose disease is resistant to 5-FU. Current studies with CPT-11 in colorectal cancer Because 5-FU and CPT-11 each have substantial single agent activity and appear to be relatively non-crossresistant, investigators have begun to examine ways in which both therapies may be used in an integrated fashion as front-line systemic therapy for patients with advanced colorectal cancer. A variety of doses and schedules have been evaluated, ranging from weekly CPT-11 administered prior to infusional 5-FU to concurrent administration of weekly CPT-11, 5FU, and leucovorin, to administration of CPT-11 and 5-FU in alternating cycles [67, 68, 79-82]. Response rates of 17% 62% have been observed in these phase I/II and phase II trials and phase III trials have begun in which the impact of this combination therapy will be compared to monotherapy with CPT-11 or 5-FU/LV. These studies should help to define the impact of CPT-11 on time to treatment failure and survival in patients with metastatic colorectal cancer. Other solid tumours Publications reporting the results of phase II studies in which CPT-11 was used as a single agent to treat other solid tumour types are shown in Table 3. Response rates of ll%-36% have been reported in previously untreated patients with [83-87]. CPT-11 appears to be quite active against small cell lung cancer (SCLC) with response rates of 37%-47% reported in previously untreated patients and 16% in patients who had progressed after responding to one prior chemotherapy regimen [88, 89]. CPT-11 has moderate activity against pancreatic cancer with response rates of approximately 10% [90, 91]. Three preliminary reports [92-94] suggest that CPT-11 is an active agent against squamous cell carcinoma of the cervix although a fourth is negative [95]. Individual studies of CPT-11 against ovarian, gastric, skin, and breast cancer, and lymphoma suggest that single-agent CPT-11 may also have activity against these malignancies [96-101]. Preliminary reports on phase II studies of CPT-11- based combinations have begun to appear. The CPT-11 + cisplatin combination appears quite promising for patients with previously untreated. Response rates in the range of 31% 54% have been reported using a variety of schedules [ ]. CPT-11 has also been used in combination with etoposide [65], or carboplatin (CBCDA) [105] to treat and these combinations also appear to have activity in this disease (Table 4). High response rates have been achieved using the CPT-11 + cisplatin combination against SCLC, gastric, and ovarian cancers [101, ] and warrant further study. There is a preliminary report of a combination of CPT-11 and mitomycin C (MMC) in clear cell carcinoma of the ovary [109]. Given the profound suppressive effect of CPT-11 on blood lymphocytes, it has been evaluated in patients with relapsed lymphoma. The combination of CPT-11 and adriamycin has demonstrated promising activity in patients with lymphoma who had relapsed after adriamycin-based therapy [110]. Toxic ity The main toxicities observed with CPT-11 are an acute cholinergic-like syndrome, delayed onset diarrhoea, neutropenia, nausea and vomiting, fatigue and alopecia. The early-onset cholinergic-like syndrome appears to be due to inhibition of anticholinesterase by CPT-11. It produces symptoms during or shortly after drug infusion which include diarrhoea, diaphoresis and abdominal cramps and has been reported to occur in up to 79% of patients [111]. Hyperlacrimation and rhinorrhoea are also observed. These effects are short-lasting and respond within minutes to atropine 0.25 to 1.0 mg intravenously or subcutaneously. This is not a DLT and patients who experience the cholinergic reaction can receive prophylactic atropine prior to receiving additional cycles of CPT-11. Delayed onset diarrhoea of any grade occurs in up to 87% of patients and affects approximately 60% of treatment cycles. It is severe (WHO or NCI CTC grade 3 to 4) in up to 37% of patients and complicates approximately 12% of all treatment cycles. The onset is usually between days 5 to 12 of the treatment cycle [112]. While the median duration of diarrhoeal episodes is two to five days, grade 3 to 4 diarrhoea tends to last, on average, five to seven days. Recent animal studies have shown that treatment with CPT-11 induced epithelial vacuolation and apoptosis in the ileum, and goblet-cell hyperplasia with excessive production of mucin in the caecum [113]. The functional correlates of these structural changes would be malabsorption of water and electrolytes and hypersecretion of rectal mucin, respectively, resulting in a secretory/exudative diarrhoea. Grade 3 or 4 neutropenia occurs in 26% to 46% of patients and accompanies 12% to 20% of all treatment cycles. Because of the short duration of neutropenia, the incidence of neutropenic fevers is low (less than 5%). The combination of grade 4 neutropenia and grade 3 to 4 diarrhoea can be dangerous, and requires aggressive supportive care. Some degree of nausea or vomiting is experienced in 71% to 86% of patients. Grade 3 to 4

6 842 Table 3. Phase II studies with CPT-11 in other solid tumour types; single-agent therapy. Tumour type CPT-11 dosage n entered; evaluable Responses Reference Pancreatic Pancreatic SCLC SCLC SCLC Gastric Breast Breast Cervical Cervical Cervical Cervical Skin Squamous Melanoma Ovarian Ovarian NK Prior Rx NK CDDP-based CDDP + ETOP Permitted Permitted One prior CT CDDP-based NK CDDP-based Permitted Permitted 350 mg/m 2 q3w 150 mg/m 2 q2w 350 mg/m 2 q3w 350 mg/m 2 q3w 350 mg/m 2 q3w 150 mg/m 2 q2w mg/m 2 qlw 350 mg/m 2 q3w 350 mg/m 2 q3w 125 mg/m 2 qlw 150 mg/m 2 q2w 125 mg/m 2 qlw or 150 mg/m 2 q2w or 150 mg/m 2 q2w 34; 32 61; 35 19; 11 31;27 73; 72 67; 67 26; 26 NK;41 35; 35 16; 15 32; 32 81;6O 79; 65 29; 12 42; 34 46;42 69; 55 16; 14 42; 33 38; 32 75; 55 27;27 25;25 3/32 (9.4%) 4/35(11.4%) 4/11(36.4%) 3/27(11.1%) 23/72(31.9%) 23/67(34.3%) 0 6/41 (14.6%) 13/35(37.1%) 7/15(47%) 5/32(16%) 14/60 (23.3%) 15/65(23.0%) 1/12(8.3%) 5/34(14.7%) 9/42(21.4%) 13/55(23.6%) 0 13/33(39%) 3/32(9%) 13/55 (23.6%) 8/27(29.6%) 4/25(16.0%) [90] [91] [83] [84] [85] [86] [87] [86] [88] [89] [96] [97] [98] [92] [93] [94] [95] [99] [100] [101] Abbreviations: NK - not known; CT - chemotherapy; - non-small-cell lung cancer; SCLC - small-cell lung cancer; CDDP - cisplatin; ETOP - etoposide (VP-16). vomiting occurs in 12% to 20% of patients, but is manageable with 5-HT3 antagonists and dose reduction. Other common toxicities have included fatigue or asthenia in 76% to 80% of patients (severe in 12% to 20%) and alopecia (usually partial) in 60% to 81% of patients. There have been case reports of dyspnea and interstitial pneumonitis occurring in patients treated with CPT- 11 [85]. It appears that this toxicity is rare and probably represents an idiosyncratic reaction to CPT-11. Nevertheless, patients who report shortness of breath while receiving CPT-11 should be evaluated carefully to determine the aetiology for this symptom. Management of toxicity The severity of the delayed-onset diarrhoea with CPT-11 has been substantially reduced through the use of an intensive schedule of loperamide as originally described by Abigerges et al. [41]. This intervention allowed French investigators to reach doses as high as 750 mg/ m 2 every three weeks before encountering dose-limiting neutropenia. American investigators reported a substantial reduction in the frequency of severe diarrhoea, with grade 3 to 4 diarrhoea falling from 24% to 9% (data on file, Pharmacia & Upjohn) after adoption of this regimen. Dose reductions taken during the four weeks of treatment also contribute to controlling this toxicity in the weekly schedule. French investigators have had promising results with the antisecretory enkephalinase inhibitor acetorphan used in combination with loperamide, and this strategy is being evaluated in randomized studies [114]. An attempt to prevent the occurrence of diarrhoea through the use of prophylactic acetorphan and loperamide was unsuccessful and proved no better than waiting until the diarrhoea began before initiating treatment [115]. Attempts to establish pharmacodynamic relationships between CPT-11, its metabolites, and delayed diarrhoea have been undertaken. While some groups have identified correlations between SN-38 AUC or biliary index and diarrhoea, these correlations have not been consistent across studies, and have yet to be independently validated. This may reflect the complex pharmacology of CPT-11 and the presence of a number of recently identified metabolites that may be important mediators of normal tissue toxicity (and potentially cytotoxicity) of this compound [38,40,42,44,116,117]. There are little data in the literature regarding the routine use of G-CSF to offset neutropenia in patients treated with single agent CPT-11. Studies have utilized G-CSF in an attempt to increase the dose-intensity of CPT-11 using the weekly schedule [118]. The maximum tolerated dose (MTD) of CPT-11 was 145 mg/m 2 with G-CSF, which represents a 16% increase in the MTD of

7 843 Table 4. Phase II studies with CPT-11 in other solid tumour types; combination therapy. Tumour type Study treatment n entered; evaluable Responses Reference SCLC Gastric Gastric Ovary (clear cell) Ovary Lymphoma No platinum NK ADR CPT-11 +VP-16 + G-CSF CPT-11 +CDDP CPT-11+CDDP CPT-11 + Infus. CDDP + G-CSF CPT-11 +CBDCA CPT-11 +CDDP CPT-11 +CDDP CPT-11+CDDP CPT-11+MMC CPT-11+CDDP CPT-11 +ADR 61; 59 70; 69 32; 32 52; 52 41;41 12; 10 75; 32 24; 24 44; 44 12; 7 25; 20 12; 12 13/59(22.0%) 33/69(47.8%) 8/32(25.0%) 16/52(30.8%) 22/41 (53.7%) 4/10(40.0%) 25/32(78.1%) 10/24(41.7%) 21/44(48%) 5/7(71.4%) 8/20(40.0%) 4/12(33 3%) [65] [102] [103] [104] [105]" [106] [107] [108] [109] [101] [98] (N)SCLC - (non)-small-cell lung cancer; CT - chemotherapy; ADR - adriamycin-containing regimen; NK - not known. * Phase I/II study with dose escalation. CPT-11 alone. The clinical implications of this slight increase in CPT-11 dose have not yet been explored. Future directions for CPT-11 Further investigations of alternative schedules of administration of CPT-11, such as continuous infusion or every other week bolus dosing, are underway or planned. Studies have begun to evaluate doses of 250 mg/m 2 every two weeks or 500 mg/m 2 every three weeks in settings where patients can be closely monitored. As noted above, CPT-11 is also being studied in combination with 5-FU as first-line treatment for advanced colorectal cancer. There is a great deal of activity focused on exploration of CPT-11-based drug combinations. These trials will evaluate CPT-11 with other thymidylate synthase inhibitors, taxanes, alkylating agents, platinums, and mitomycin-c. The oral route of administration for CPT-11 has proven feasible and may represent one way of exposing tumours to low concentrations of drug over a sustained period of time [48]. Preclinical studies have also demonstrated that CPT-11 possesses radiosensitizing effects against selected lung cancer cell lines, and early phase II clinical trial results appear to confirm this with one study reporting a 79% response rate in patients with stage IIIA and IIIB [119, 120]. Much still needs to be done to realize the full clinical impact of CPT-11. Optimal utilization of CPT-11 in patients with metastatic colorectal cancer will require evaluation of CPT-11 as part of a front-line treatment regimen, initially with 5-FU administered in a variety of schedules, doses, with or without leucovorin. The impact of CPT-11 in earlier stages of disease as adjuvant therapy has yet to be explored. The response rates for CPT-11 + cisplatin combinations in patients with smallcell or non-small-cell lung cancer appear quite promising from phase I and II trials, but will require more definitive evaluation in phase III trials. Exploratory phase II trials are also in development in other solid tumours in which preclinical activity has been observed, for example, sarcoma and mesothelioma. Topotecan Pharmacokinetic studies In pharmacokinetic studies, the half-life of topotecan varies from 1.7 to 3.0 hours when administered daily for five days once every three weeks to 2.4 to 8.0 hours when administered as a 24-hour infusion [ ]. Typical clearance on the five-day schedule is /min/m 2 and the 48-hour urinary excretion is between 20% and 80% [125]. There is some evidence that myelosuppression with topotecan is correlated with AUC and peak plasma concentration although antitumour activity may not be; the outcome of large population studies is awaited [125, 126]. Phase I studies Phase I studies have tested a wide variety of schedules including 30-minute intravenous infusions daily x 5 [122, 127, 128], 24-hour infusions administered weekly [129, 130], 72-hour infusion administered once every three weeks [131], a 120-hour infusion every three to four weeks [132], and a 21-day continuous infusion [133]. These schedules were chosen after preclinical data suggested that more protracted exposure to topotecan might optimize the chances that sufficient drug concentrations would be present during the S-phase of the cell cycle. The DLT with all schedules is neutropenia with or without thrombocytopenia. Dose intensity with the three-week continuous infusion exceeded that with other schedules but this was associated with a higher incidence of thrombocytopenia and cumulative anaemia [133, 134]. Phase I studies of oral administration include

8 844 daily dosing x 5 or 10 days every three weeks [135, 136]. Due to the development of grade 4 thrombocytopenia, the addition of G-CSF was not successful in increasing the MTD of topotecan using the standard daily x 5 dosing schedule [130]. In a 120-hour continuous infusion schedule tested in children with acute leukaemia, the DLT was mucositis [137]. Phase II studies Responses were noted during phase I studies of topotecan in small-cell and non-small-cell lung cancer, ovarian cancer, oesophageal cancer, and colorectal carcinoma, which prompted subsequent phase II studies. Additionally, minor responses were noted in renal cell carcinoma and squamous cell carcinoma of the skin [54], Most of the phase II studies published to date have used a schedule of topotecan 1.5 mg/m 2 daily x 5 consecutive days with cycles repeated every 21 days. The early clinical development of topotecan has focused on ovarian and small-cell lung cancers. Several single centre and one European multicentre phase II trials demonstrated consistent activity for topotecan as second-line treatment for patients with ovarian cancer who had already received a platinum agent [ ] (Table 5). Objective response rates ranged from 14% to 25% and the responses lasted a median of 5.4 to 8.9 months. This prompted a randomized trial in which topotecan was compared to paclitaxel in patients who had developed progressive disease after having received one platinum-based regimen [141]. While no signficant differences were detected in objective response rate or median survival, patients treated with topotecan had a significantly longer time to tumour progression than those treated with paclitaxel (23 vs. 14 weeks, respectively, P = 0.002). Haematological toxicities were more common in the topotecan-treated patients, but these toxicities were reversible and non-cumulative. This data supports the notion that topotecan is a very active drug in this setting. Since most patients in the US and Europe now receive a platinum and taxane as front-line chemotherapy for advanced ovarian cancer, evaluation of the activity of topotecan in this setting would be quite informative. Recently, a phase II trial of topotecan was reported in patients who had progressed within six months of receiving a platinum + paclitaxel combination [142]. An objective response rate of 12.9% and a median progression-free survival of 17 weeks were reported, indicating that topotecan has important activity in this setting, as well. Phase II data for topotecan in ovarian and other solid tumors is summarised in Table 5. In addition to ovarian cancer, topotecan shows a promising response rate in SCLC either as first-line chemotherapy (RR = 39%) or as salvage chemotherapy (RR = 6%-38%) [ ]. Approximately 30% of topotecan crosses the bloodbrain barrier and can be detected in the cerebrospinal fluid. This may be one reason why topotecan has demonstrated particular activity against brain metastases from SCLC. Preliminary results from a trial that included 16 such patients report four complete and six partial responses (central nervous system response rate 63%) [147, 148]. There is also evidence that topotecan is active in previously untreated [ ] although this has not been confirmed in all studies [152]. There is early evidence of activity in squamous cell carcinoma of the head and neck [153] and soft tissue sarcoma [154], and limited activity in prostate carcinoma [155, 156]. Topotecan has only modest or no activity in recurrent gliomas [157, 158], chronic lymphocytic leukaemia [159], renal cell carcinoma [160], pancreatic adenocarcinoma [161], cisplatin-refractory germ cell tumours [162], soft tissue sarcoma, previously-treated urothelial tumors [163], breast cancer [ ] or relapsed paediatric brain tumours [167]. Topotecan does not appear to have substantial activity against previously untreated colorectal cancer, either when given on a standard daily x 5 schedule [168, 169] or by a 21-day continuous infusion [134,170,171]. Toxic ity In general, the pattern of toxicity observed in phase II studies with the five-day regimen of topotecan reflects that predicted on the basis of the phase I data [172], Phase II studies report grade 3 or 4 neutropenia accompanying 30% to 80% of treatment cycles [138, 155, 160]. Grade 3 or 4 thrombocytopenia has been reported in 10% to 15% of cycles [138, 144] and may be sustained [150]. Febrile neutropenia has been reported in 10%- 25% of patients [139, 140]. The incidence of febrile neutropenia has decreased with the use of G-CSF. Grade 3 and 4 diarrhoea or vomiting have been reported in a small proportion of patients and these toxicities appear to be less frequent with topotecan than with CPT-11 [149]. Other adverse effects, such as anaemia, have generally been mild and easily controlled, although anaemia may be cumulative with the three-week continuous infusion schedule [134]. Neutropenia may not be alleviated by G-CSF administered on a conventional schedule [54]. However, in a study of previously untreated patients with renal cell carcinoma or melanoma who received topotecan 1.5 mg/m 2 daily for five days, grade 4 neutropenia occurred in significantly fewer patients who received GM-CSF 250 ug/m 2 twice daily for five days prior to topotecan in addition to the conventional 250 ug/m 2 daily for seven days following topotecan (3/11, 27% vs. 10/13, 77%; P = 0.02) [173]. Current clinical trials A phase III trial is underway comparing topotecan to cyclophosphamide, doxorubicin and vincristine [CAV] as second-line therapy in patients with SCLC and progressive disease following one platinum-based chemo-

9 845 Table 5. Phase II studies with topotecan in solid tumours; single-agent therapy. Tumour type Regimen n entered; evaluable Responses Reference Ovarian Ovarian Ovarian^ Ovarian SCLC SCLC SCLC SCLC (squamous) Head & neck Glioma Ghoma ST sarcoma Prostate CRC CRC CRC Breast Breast Breast Pancreatic Renal Germ cell Bladder 1 prior platinum < 2 prior CT _ Platinum Paclitaxel and platinum CDDP + VP-16 'Sensitive' 'Refractory 1 1 prior CT Adjuvant NK NK Cisplatin 1,5 m /m 2 /day x 5_q3w 1.5mg/m 2 /day x 5 q3w 1.25mg/m 2 /day x mg/m 2 /day x 5 q3w + G-CSF 1.3 mg/m 2 /day x 3 q4w 1.5mg/m 2 /day x 5 q3w 2.0 mg/m 2 /day x 5 q3w 0.4 mg/m 2 CIV x 21 days q4w mg/m 2 /day CIV x 21 days q4w 0.5 mg/m 2 /day CIV x 21 days q4w 22.5 mg/m 2 q 3 wkor 1.5 mg/m 2 24 CIV qwk NK 111; 92 20; 16 30; 28 62; 62 32; 28 46,45 47; 47 95; 74 48; 48 38; 38 40; 37 43; 37 20; 20 35; 29 18; 15 31; 31 29; 26 19; 16 34; 28 42; 40 26; 22 19; NK 16; 14 20; 20 18; 18 16; 16 15; NK 15; 14 15; 15 46; 42 15/92(16.3%) 4/16(25.0%) 4/ %) 8/62 (12.9%) " 3/28(11.0%) 17/45(37.8%) 3/47(6.4%) 8/74(10.8%) 19/48(39.6%) 7/38 (18.4%) 3/37(8.1%) 5/37(13.5%) 0 7/29(24.1%) 4/15(26.7%) 3/31 (6.5%) 3/26(11.5%) 2/16(12.5%) 2/28(7.1%) 4/40(10%) 2/22(9.1%) 0 5/14(35.7%) 2/20(10.0%) 1/18(5.6%) /42 (9.5%) [140] [138] [139] [142] [144] [145] [146] [143] [149] [150] [152] [151] [153] [157] [158] [154] [155] [171] [170] [168] [164] [165] [166] [161] [160] [162] [163] Abbreviations: (N)SCLC - (non>small-cell lung cancer; CRC - colorectal cancer; ST - soft tissue; NK - not known; CT - chemotherapy; CIV - continuous intravenous infusion; CDDP - cisplatin; VP-16 - etoposide. therapy regimen. There are no completed phase II studies of topotecan in combination with other agents, although in vitro studies suggest synergistic cytotoxicity for topotecan and CDDP in human tumour cell lines, which is most pronounced when CDDP is administered first [174]. Topotecan is being evaluated in phase I studies in combination with paclitaxel [175], CDDP [176, 177], cyclophosphamide [178], and doxorubicin [179]. Future direction/or the development of topotecan Additional large studies in patients with ovarian cancer are planned that will determine the contribution of topotecan when used as part of front-line, multiagent chemotherapy. Given the substantial myelosuppressive effect of topotecan and the other drugs used in this setting, one of the biggest challenges will be to devise a way in which all drugs may be given in adequate doses without inducing unacceptably severe or prolonged neutropenia. Topotecan, in common with many of the other camptothecins under clinical development, has significant oral bioavailability [180]. The oral route of dosing may provide a convenient way of achieving prolonged exposure to the drug without the inconvenience of ambulatory infusion pumps and semipermanent catheters. A phase I trial evaluating 5-, 10-, and 21-day oral dosing schedules has been performed [135,136]. This may be the preferred route of administration for topotecan in those situations in which protracted use may be desirable, such as in the setting of radiation therapy. Because of the potential for topo I inhibitors to lead to upregulated expression of topoisomerase II, sequential administration of topotecan and etoposide has been explored [181]. Preliminary results from a phase I study in which serial measurements of tumour topoisomerase were made in patients receiving topotecan on days 1 to 3 and etoposide on days 7 to 9 suggest that this measurement technique is feasible and may help to determine optimal scheduling of topoisomerase inhibitor combinations [182]. Preclinical evidence suggests that topotecan may act synergistically with radiation by inhibiting radiation damage repair; cytotoxicity of topotecan is enhanced during the 30 minutes following irradiation [183,184]. A published phase I study of topotecan plus radiotherapy for [185] shows the combination may be used safely; further studies are needed.

10 846 9-Aminocamptothecin (9-AC) Although preclinical data showed 9-AC to have greater activity than camptothecin against human tumour xenografts, including Lewis lung carcinoma and B16 melanoma [22], its clinical development has been hampered by its relatively poor solubility in water. More recently, a colloidal dispersion (CD) preparation of 9-AC has become available and has entered phase II trials. It has virtually the same pharmacokinetic and toxicity profile as the original preparation, but is easier to administer clinically. Prolonged exposure to 9-AC appears to be essential for antitumour activity. As a result, due to its short terminal half-life, clinical trials of 9-AC have relied primarily on prolonged intravenous infusions ranging from 72 hours to 21 days [13, 186]. Less than 10% of the drug circulates in plasma as the active lactone form [186]. Preclinical studies suggest that the combination of cisplatin and 9-AC may be synergistic, due, at least in part, to the ability of 9-AC to inhibit the reversal of cisplatin-induced DNA damage [187]. In phase I studies in which 9-AC was administered as a 72-hour continuous infusion repeated once every two to three weeks, the MTD was ug/m 2 /hour (47 to 59 ug/m 2 /hour with G-CSF) and neutropenia was the DLT [12, 188]. Thrombocytopenia was also prominent. Minor responses were seen in patients with gastric, colon and NSCL cancers. Other dose-finding and pharmacokinetic studies are underway, including an investigation of continuous infusion over 21 days [189]. Additional toxicities reported to have been observed in phase I studies include anaemia, nausea, vomiting and diarrhoea [13]. Early reports from phase II studies have begun to appear. Responses have been observed in patients with previously untreated colorectal cancer, previously-treated breast cancer, and relapsed lymphoma [ ]. GG-211 (GI ) GG-211 is a water-soluble camptothecin analogue found to be comparable in potency to topotecan in vitro, and active in cell cultures of HT-29 and SW-48 colon cancer, and in MX-1 and PC-3 xenografts [193]. Two regimens have been tested [14]: a 30-minute infusion daily for five days every three weeks, and a 72-hour infusion once every three weeks. Myelosuppression is dose-limiting with either schedule. Neutropenia predominates in the intermittent dosing schedules and thrombocytopenia is more prominent with long infusions [194]. The MTD of the daily x 5 schedule was 1.2 mg/m 2 /day and the DLT were neutropenia and thrombocytopenia. Haematological nadirs occurred around day 15 and tended to be protracted, lasting a median of 7 to 10 days. As a result, six of the 15 courses administered at the MTD had to be delayed to allow adequate recovery of blood counts. Other toxicities were mild and infrequent and included nausea, vomiting, headache and alopecia. No diarrhoea was observed. The terminal half-life of GG-211 was 3.7 ± 1.2 hours. One partial remission, lasting six weeks, was observed in a patient with recurrent colorectal cancer [195]. Responses have been observed in patients with colon, breast, ovary and liver cancers. Several preliminary reports of phase II studies utilizing the 1.2 mg/m 2 /day qd x 5 schedule have appeared recently. In patients with chemotherapy naive, locally advanced or metastatic, topotecan had only modest activity, with 2 of 22 patients achieving a partial response (RR = 9%) [196]. In patients with ovarian cancer that had recurred 4 12 months following platinum chemotherapy, 5 of 23 patients responded (RR = 22%), including one patient with a complete response [197]. Slightly less activity was observed in patients with a treatment-free interval of less than four months. Further phase I and II studies are in progress, and an oral formulation is being evaluated [198]. DX-8951f DX-8951f is a water-soluble camptothecin analogue that, in vitro, is the most potent topo I inhibitor tested to date [199]. In tests against cultures of the human lung cancer cell line PC-6, DX-8951f wasfivetimes as potent as SN-38 and ten times as potent as topotecan. Cultures of the SN-38-resistant subline PC-6/SN2-5 were ten times as resistant as the parent line to SN-38 and topotecan but were not resistant to DX-8951f. DX-8951f has recently entered phase I clinical trials that will evaluate bolus (q 3 week) and well as repeated (qd x 5, weekly, and continuous infusion) schedules. Oral camptothecin Orally-administered camptothecin is still being used in its traditional formulation in the Republic of China, and phase I studies have been performed using a daily oral dosing schedule in the US [200]. Given once daily x 21 followed by a seven-day rest, DLT was achieved at the 15.4 mg/m 2 /day dose level of 20(S)-camptothecin. This took the form of diarrhoea, but neutropenia and chemical cystitis were also observed. Oral absorption was rapid with peak plasma levels achieved within two to four hours. The MTD was 8.7 mg/m 2 /day. Although antitumour activity was noted and partial responses observed in patients with breast or prostate cancer or malignant melanoma, this compound is not undergoing further testing at the present time, in part due to the development of other oral camptothecin analogues. 9-Nitrocamptothecin (9-NC) 9-Nitrocamptothecin is actually an oral prodrug that is rapidly converted to 9-aminocamptothecin following administration [201]. A phase I clinical trial of 9-NC

11 847 was conducted using afive-dayon/two-day off schedule [202]. Myelosuppression was dose limiting, but chemical cystitis was also observed in 36% of patients. Responses have been observed in patients with pancreatic, ovarian and breast cancers as well as in a patient with metastatic cholangiocarcinoma. Early results from phase II trials have begun to appear. In a group of women with heavily-pretreated ovarian cancer, 2 of 25 patients who received 9-NC 1.5 mg/m 2 /day for four consecutive days each week achieved a partial response [203]. Mechanisms of resistance to camptothecins Development of cellular resistance to camptothecin analogues appears to be a multifactorial phenomenon [204]. The principal mechanisms thought to play a role in resistance to camptothecins are summarized below. Overexpression of MDR1 A number of drug-resistant tumour cell lines overexpress the multidrug resistance gene MDR1 and its protein product, p-glycoprotein (pgp). Increased levels of pgp result in increased efflux of the drug from the cell and decreased intracellular accumulation of the drug [17, 36, 205]. Topotecan can serve as a substrate for pgp, but the degree of cross-resistance conferred by overexpression of pgp is relatively low, suggesting that there are other important drug resistance mechanisms involved [206]. The data regarding the effect of the p-glycoprotein pump on CPT-11 are somewhat limited, but it appears that the resistance against CPT-11 in pgpexpressing cells is greater than that for SN-38 [207] Reduced topoisomerase I levels Certain tumour cell lines resistant to camptothecin analogues contain reduced topo I levels [29, 208, 209]. It is thought that low levels of topo I reduces the target available for blockade by topo I inhibitors and decreases the sensitivity of the cell to agents with this mechanism of action. Unfortunately, this phenomenon has been difficult to demonstrate clinically. In one series, no correlation was found between bone marrow mononuclear cell topo I content and clinical response to topotecan or sensitivity of leukaemia cells to topotecan ex vivo, despite as much as an eight-fold difference in topo I content between patient samples [210]. Another hypothesis is that the cell's ability to downregulate topo I should correlate with resistance to topo I inhibitors. Indeed, transient and rapid reduction in topo I levels has been demonstrated to occur in clinical samples or peripheral blood mononuclear cells (PBMQ following exposure to camptothecin analogues in vitro and in vivo [ ]. However, neither the baseline PBMC topo I levels nor the relative decrease of topo I levels from baseline correlated with clinical response to therapy. In some cases, a transient increase in topoisomerase II can compensate for the loss of topo I function. This phenomenon has been demonstrated to occur in human tumours following exposure to topotecan [214] or camptothecin [215] and provides a rationale for sequential therapy with topo I and II inhibitors. Some have observed changes in topo I activity without changes in topo_ I expression. This suggests that the post-transcriptional events, such as phosphorylation or poly-adp ribosylation, could have a significant impact on the activity of topo I and on its susceptibility to inhibition [216]. Subcellular redistribution Topoisomerase I is synthesized in the cytoplasm and must enter the nucleus and bind to DNA that is undergoing replication and transcription in order to exert its function. In addition, some topo I may be associated with DNA that is not actively replicating or being transcribed. Therefore, measurement of total cellular topo I may be misleading and may account, in part, for the lack of clinical correlation between levels of topo I expression and sensitivity to damage from topo I inhibitors. Several investigators have described a subcellular redistribution of topo I from the nucleoli to other parts of the nucleus or to the cytoplasm following exposure of the cell to camptothecin analogues. The significance of thesefindingswith regard to cellular sensitivity to camptothecin analogs is under active investigation [217, 218]. Mutant topoisomerase I Some tumour cells that are resistant to camptothecin contain a mutant form of topo I [219]. One type of mutation, such as that which occurs through the substitution of glycine for aspartic acid at positions 533 and 583, results in decreased topo I function. A second type of mutation, substitution of serine for phenylalanine at position 361, results in an enzyme that has normal catalytic activity but reduced DNA binding affinity. Since camptothecins bind to the topo I-DNA cleavable complex, rather than to the enzyme itself, impairment in cleavable complex formation can also result in camptothecin resistance [220]. While mutational analysis has provided significant insights into the relationship between DNA, topo I, and topo I inhibitors, the frequency and relevance of these alterations to the resistance that occurs in clinical specimens remains to be explored. Impaired drug activation While most camptothecin analogues have a direct inhibitory effect on topo I function, CPT-11 must first undergo metabolic activation by endogenous carboxylesterases. Cell lines that lack carboxylesterase activity, and therefore are unable to convert CPT-11 to SN-38, demonstrate resistance to this camptothecin analogue [221]. In a preliminary report, there appeared to be an association between carboxylesterase activity and

12 848 sensitivity to CPT-11 in fresh human tumour samples [222]. Alterations in the cell cycle A characteristic of some cells that are resistant to topoisomerase inhibitors is a lengthened cell cycle [223, 224]. This presumably has the effect of reducing the proportion of cell in S-phase at any given time, thereby limiting the proportion of cells that would incur DNA damage from the camptothecins. Cell cycle arrest in G1/G2 or G2/M is another way in which cells may become resistant to the camptothecins. In vitro, strategies that 'drive' cells through to S-phase have been successful in restoring sensitivity of the cells to camptothecin [225, 226] Altered downstream events The ultimate effect of any drug on the cell appears to depend upon the ability of the cell to 'recognize' that damage and to mobilize systems that will either repair the damage or force the cell to undergo apoptosis. These 'downstream events' involve signal transduction elements, such as PKC-alpha and tumour suppressor genes that regulate cell cycle checkpoints, such as cyclin Bl, p53, bax, bcl-2, bcl-x, p21 and cdc-2, among others [ ]. Efforts to correlate the expression of a single cell cycle regulatory gene with sensitivity or resistance to camptothecins have been largely unsuccessful [230]. It is more likely that measurement of the relative expression of multiple regulatory genes will prove to be the most useful correlate of camptothecin sensitivity. Although normal p53 status has been implicated in apoptosis that occurs following exposure to various types of cellular damage, normal p53 does not appear to be necessary or sufficient for a cell to undergo apoptosis following exposure to camptothecins. Cells without functional p53 can undergo apoptosis following exposure to a variety of topo I and II poisons [231, 232]. Camptothecin, among other DNA-damaging agents, increases wild-typep53 levels in damaged cells [233]. Experiments using cell-free systems suggest that soluble mediators of apoptosis are generated in cells exposed to camptothecin [232]. Serine proteases, such as granzyme B or cysteine protease, have been proposed as putative mediators of apoptosis since serine protease inhibitors can effectively abrogate this effect. Non-camptothecin topoisomerase I inhibitors The usefulness of the camptothecin analogues as a means of treating cancer has led to a search for other classes of drugs with the same target but different modes of action. There is now a range of other molecules that have been shown experimentally to have topo I-inhibiting properties. Antibiotics Saintopin, UCE6 and UCE1022 are structurally-related anthraquinone antibiotics that induce topo I-mediated cleavage. Saintopin also induces topoisomerase IImediated DNA cleavage, as does the indole derivative intoplicine [234]. Saintopin-resistant cell Lines show cross-resistance with CPT-11. The mechanism of crossresistance appears to be rapid down-regulation of topo I following drug exposure [235]. New naphthacenecarboxamide antibiotics designated TAN-1518 A and B inhibit topo I activity and also suppress the growth of various murine and human tumour cells in culture [236]. TAN-1518 interacts with topo I in a fashion quite distinct from camptothecin and, as a result, is active in some camptothecin-resistant cell lines. Rather than stabilizing the cleavable complex, TAN-1518 directly inhibits the catalytic activity of topo I and inhibits cleavable complex formation [237]. Indolocarbazole derivatives The semisynthetic indolocarbazole derivatives KT6006, KT6528, ED-110 and NB-506 have been shown to have toposiomerase I-mediated DNA cleavage activities, comparable with those of camptothecin, and some experimental antitumour activity [ ]. Bulgarein Bulgarein is a benzofluoranthene compound, originally isolated as a fungal pigment, which causes a distortion of the DNA helical structure by a mechanism thought to involve topo I and the cleavable complex [241], DNA minor groove binders The DNA minor groove binding ligands Hoechst dye and Hoechst dye can induce topo I-mediated DNA cleavage by a mechanism distinct from that employed by camptothecin derivatives [242]. Bulgarein is also considered to have some DNA minor groove binding activity [243]. Plant extracts The benzophenanthridine alkaloid plant extracts nitidine and fagaronine and the berberine-type alkaloid extract epiberberine have been shown to induce topo I-mediated DNA cleavage [243, 244]. Coralyne, a berberine extract, is one of the most active topo I inhibitors of this class. In vitro, coralyne and nitidine have demonstrated activity against camptothecin-resistant human tumour cell Lines [217]. Beta-lapachone is a plant extract that inhibits topo I by a mechanism that does not involve stabilization of the cleavable complex. It probably has a direct interaction with the enzyme itself [246]. BC-4-1 is a potent inhibitor of topoisomerases I and

13 849 II isolated from the traditional Chinese herbal medicine Tian-Shian-Wan [247]. tine Smart and Claire Viguier-Petit in the preparation of this manuscript. Drug-radiation interactions The interaction between camptothecin and radiation appears to be complex. While several studies have demonstrated synergy in vitro, others have shown less dramatic interactions ranging from sub-additivity to antagonism. Factors influencing the nature of this interaction appear to include the sequence and interval between treatments, cell cycle distribution and growth phase of the cultures, topo I expression levels, and the DNA repair capability of the cell lines used [248, 249]. The strategy of combining camptothecin analogues and radiation is just beginning to be explored clinically. Conclusions Topoisomerase I inhibitors are likely to have an increasing impact in cancer chemotherapy over the next few years. At present, all of the specific topo I inhibitors in development are derivatives of camptothecin. CPT-11 and topotecan have already reached the commercial market in most countries and have been brought forward for somewhat different indications: colorectal cancer for CPT-11 (with broader indications in Japan, including small-cell and non-small-cell lung, cervical, ovarian, gastric, breast, and skin cancers as well as lymphoma) and ovarian cancer for topotecan. The current developmental approach to these drugs centres around the development of more active drug combinations for use in front-line therapy and exploration of new schedules or routes of drug administration. Some of the drugs more recently introduced into clinical trials, such as 9-AC, 9-NC, GG-211, and DX-8951f are still undergoing broad phase II single-agent testing. The non-camptothecin inhibitors of topo I are all at the preclinical stage of evaluation. theless, there are several compounds belonging to various chemical classes which show promising experimental activity and may prove suitable for clinical development. Laboratory studies directed at understanding the extent, and limitations, of the clinical activity of these compounds are progressing at a very rapid pace. Further insight into the mechanisms responsible for clinical sensitivity and resistance to topo I inhibitors will help clinicians apply these agents in the most effective manner possible. The next five years promise to be a very exciting time with regards to our understanding and utilization of this novel class of cytotoxic agents. Acknowledgements The author would like to acknowledge the invaluable assistance of Bettina Bragas and Mike Matthews, Chris- References J_ Wang JC DNA_ topoisomerases. Annuitev Biochem 1985; 54: _ Wall ME, Wani MC, Cooke CE et al. 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