Antifolates targeted specifically to the folate receptor

Advanced Drug Delivery Reviews 56 (2004) 1111 – 1125 www.elsevier.com/locate/addr

Antifolates targeted specifically to the folate receptor Ann L. Jackman *, Davinder S. Theti, David D. Gibbs Section of Medicine, Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey, SM2 5NG, UK Received 3 October 2003; accepted 5 January 2004

Abstract Most antifolate drugs are efficiently transported by the reduced-folate carrier (RFC). However, several also bind with high affinity to the a-isoform of the folate receptor (a-FR) and there is evidence to suggest that this transport mechanism may contribute to their activity when the receptor is highly overexpressed or when the extracellular folate concentration is very low. In particular, the presence of the a-FR on tumour cell lines sensitises them to brief exposures to ZD9331. Nevertheless, it is the ubiquitous expression of the RFC in normal tissues that reduces patient tolerability to antifolate drugs. The overexpression of the a-FR in some epithelial tumours and its restricted distribution in normal tissues suggests an opportunity for the development of antifolates specifically targeted at a-FR overexpressing tumours. Potent cyclopenta[ g]quinazoline-based inhibitors of thymidylate synthase (TS) have been discovered with high and low affinity for the a-FR and RFC, respectively. This class of agent is represented by CB300638 (TS Ki = 0.24 nM) that displays high potency (IC50 f 3 nM) for A431-FBP cells (transfected with the a-FR) and KB cells (constitutive overexpression). Importantly, this activity is f 300-fold higher than for a-FR negative cell lines such as A431. In mice bearing the KB tumour xenograft we have demonstrated localisation of CB300638 to tumour and, more importantly, specific inhibition of TS in tumour and not in normal tissues. Data supports the clinical development of this class of agent with the prediction that toxicity would be reduced compared with conventional antifolate drugs. There are a number of challenges to this development posed by the uniqueness of the compounds and these are discussed. D 2004 Elsevier B.V. All rights reserved. Keywords: a-Folate receptor; Thymidylate synthase; Antifolate

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . Mechanisms of antifolate drug action. . . . . . . . . The role of the a-FR in the activity of antifolate drugs 3.1. Methotrexate . . . . . . . . . . . . . . . . 3.2. Lometrexol . . . . . . . . . . . . . . . . . 3.3. Raltitrexed, pemetrexed and ZD9331 . . . . .

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* Corresponding author. E-mail address: [email protected] (A.L. Jackman). 0169-409X/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2004.01.003

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Targeting novel antifolate TS inhibitors to tumours via the a-FR . . . . . . . . 4.1. Compound design . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. Inhibition of isolated TS . . . . . . . . . . . . . . . . . . . 4.1.2. Affinity for the folate transporters . . . . . . . . . . . . . . 4.1.3. Growth inhibition in mouse L1210 and L1210:1565 cell lines . 4.1.4. Growth inhibition in the L1210-FBP cell line . . . . . . . . . 4.1.5. Growth inhibition in human A431, A431-FBP and KB cell lines 4.2. CB300638 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1. In vitro. . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2. In vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Concluding remarks and future directions . . . . . . . . . . . . . . . . . . . 5.1. a-FR expression levels . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Evaluation of toxicity and antitumour activity in mice. . . . . . . . . . 5.3. Clinical development . . . . . . . . . . . . . . . . . . . . . . . . . 6. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.

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1. Introduction

2. Mechanisms of antifolate drug action

The a-folate receptor (a-FR) is overexpressed in some epithelial tumours and has a restricted distribution in normal tissues [1 – 6]. Because it can function as a folate transporter [6– 8], it may be of use in the development of novel antifolate drugs that are selectively transported by this receptor. Antifolates targeted to the a-FR should cause very low toxicity to those normal tissues usually susceptible to antifolate action. Most classical antifolate drugs are rapidly transferred across the plasma membrane by the ubiquitously expressed reducedfolate carrier (RFC) leading to inhibition of their enzyme targets in both tumours and normal proliferating tissues [7]. Some of these antifolates have high affinity for the a-FR and evidence suggests that, under some circumstances, a-FR-mediated uptake into tumour cells may contribute to their activity [7,9,10]. Potent antifolate inhibitors of thymidylate synthase (TS) have been synthesised in which the high affinity for the a-FR has been preserved but the affinity for the RFC has been greatly reduced. Some of these a-FR targeted antifolates are transported efficiently into a-FR overexpressing tumour cells and potently inhibit their growth. This review presents the rationale for, and the development of, a-FR targeted TS inhibitors and raises a number of issues relevant to their preclinical and clinical development.

Folates are critical cofactors in a number of biochemical reactions characterised by the transfer of 1carbon groups [11]. These reactions include some essential for the de novo synthesis of purines and thymidylate. Drugs that inhibit folate-dependent enzymes are widely used in the treatment of cancer. Much of the activity of the pyrimidine-based drug, 5fluorouracil (5-FU) is ascribed to the inhibition of TS by one of its metabolites, FdUMP [12]. This leads to depletion of thymidylate and inhibition of DNA synthesis. However, 5-FU also becomes incorporated into RNA thereby affecting its function. 5-FU and its oral prodrug, capecitabine, are important in the treatment of gastrointestinal neoplasms. Methotrexate (MTX) is a folate-based drug that primarily inhibits dihydrofolate reductase leading to inhibition of de novo thymidylate and purine synthesis and consequently DNA and RNA synthesis [13]. MTX is an important component of the treatment of osteogenic sarcoma and gestational trophoblastic disease and breast cancer (in combination with other drugs). MTX is used also in prophylaxis against central nervous system relapse in acute lymphoblastic leukaemia and as maintenance therapy following remission induction in the same disease. Raltitrexed (Tomudexk; ZD1694) is a specific folate-based inhibitor of TS and used in the treatment of advanced colorectal cancer [14 –16]. Other folatebased TS inhibitors with clinical activity that are still

A.L. Jackman et al. / Advanced Drug Delivery Reviews 56 (2004) 1111–1125

being developed include ZD9331 and pemetrexed (LY231514; Alimtak) [17 – 20]. ZD9331 is a pure TS inhibitor that is not polyglutamated and pemetrexed is predominantly a TS inhibitor but inhibition of other folate-dependent targets (e.g. glycinamide ribonucleotide formyltransferase; GARFT) may be relevant for its activity. The combination of pemetrexed and cisplatin prolongs survival in mesothelioma compared with cisplatin alone, the first time a treatment has been shown to increase survival in this disease [21]. Thus, clinical data validates folate-dependent enzymes as targets for cancer chemotherapy but, in common with all cytotoxic therapy, toxic side-effects are seen. For example, a trial of adjuvant raltitrexed in colon cancer was halted because of toxic deaths due to neutropenia and diarrhea [22]. Thirty to 40% of patients treated with capecitabine have grade 3 or 4 toxicity [23,24]. In a representative Phase II trial of pemetrexed, 30 out of 46 patients experienced grade 3 or 4 toxicity [25] although it is possible that this proportion may be reduced by folate supplementation. Although the influence of these toxicities can be reduced by careful management, they reduce the usefulness of these agents. Furthermore, the doses and schedules used to minimise these toxicities may lead to sub-optimal treatment of the tumour. The main sites of antifolate toxicity are proliferating tissues such as gut and bone marrow. This probably occurs because: 1. Most antifolates are transported into cells by the ubiquitously expressed RFC. 2. Proliferating tissues are highly dependent on the enzymes that the antifolate drugs inhibit. The high overexpression of the a-FR in some epithelial tumours, notably ovarian carcinoma, offers an opportunity to design novel antifolates specifically transported by the receptor. Evidence supporting the concept comes from experimental work showing that the a-FR can operate as an antifolate transporter.

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clinical development. Studies are described that refer to cell lines cultured in either low or physiological folate concentrations. For the purposes of this review, low folate generally refers to studies in folic acid free media supplemented with 1 nM 5-formyl tetrahydrofolic acid (folinic acid; leucovorin; LV) and physiological folate concentration refers to 20 nM LV. 3.1. Methotrexate The transport of MTX into tumour cells has been studied extensively. Most transport occurs via the RFC (Km f 1 – 5 AM) a high capacity, low affinity membrane transporter [7,8]. However, MTX also binds to the a-FR although its affinity is weak (Ki f 100 nM) compared with natural folate substrates such as folic acid and its more biologically relevant cofactor form, 5-methyl tetrahydrofolic acid (0.35 and 1 nM, respectively) [26]. Two lines of evidence point to a-FR-mediated transport of MTX. First KB cells selected for MTX resistance express lower levels of FR compared with wild-type cells [27]. Second, transfection of a-FR into some [28] but not all [29] cell lines increases MTX transport or sensitivity to MTX. In addition, MTX is active in tumour cell lines that are deficient in the RFC but highly overexpress the a-FR [9,30]. These studies were performed in media containing low folate. However, the activity of MTX in tumour cell lines coexpressing the RFC and the a-FR was attributable to the more efficient RFC-mediated transport [30,31]. For example, the IC50 for the inhibition of the growth of human A431-FBP cells (transfected with the a-FR) were the same as for A431 cells (physiological or low folate concentration) (Table 1) [31]. Similarly, the IC50 for KB cells was not decreased by the coaddition of 1 AM folic acid to competitively inhibit binding to the a-FR [31]. It is unlikely that a-FRmediated transport of MTX is important in its clinical activity. 3.2. Lometrexol

3. The role of the A-FR in the activity of antifolate drugs The activity of antifolate drugs is reviewed that are used as standard treatment for cancer, or are still in

Lometrexol (DDATHF) is an inhibitor of GARFT, and therefore, inhibits the de novo synthesis of purines [32]. Clinical trials were complicated by delayed toxicity including mucositis and myelosuppression that were related to a prolonged terminal

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Table 1 Properties of antifolate drugs

Raltitrexed ZD9331 Pemetrexed Lometrexol MTX

Relative affinity for a-FR (% folic acid)a

Affinity for L1210 RFC (inhibition of 3 H MTX uptake, Ki [AM])

61 54 120 78 <1

2.5 2.1 6.0 1.8 4.0

3.3. Raltitrexed, pemetrexed and ZD9331 Inhibition of cell growth, continuous exposure IC50 (AM)b A431

A431-FBP

0.0031 0.086 0.040 0.0091 0.032

0.00073* 0.016* 0.014* 0.0024* 0.027

Results given as the mean of at least three separate experiments. a Data from Westerhof et al., 1995 [9] and Theti and Jackman [31] (a-FR expressed by L1210-FBP cells). b Theti and Jackman [31]. A431-FBP cells are transfected with the a-FR and A431 cells are neotransfected [70]. Cells grown in a physiological folate concentration (20 nM LV as folate source). Results are given as the concentration to inhibit growth by 50% compared with controls (IC50). * P < 0.05 compared with A431.

phase plasma half life, ascribed to release of compound stored in the liver as polyglutamates. Lometrexol has an affinity for the a-FR 60 –90% of folic acid [9,31] and is highly active against RFC-deficient, a-FR overexpressing L1210-FBP cells, consistent with a-FR-mediated uptake [9]. Recently, we demonstrated that the RFC proficient human A431-FBP cell line displays a 4-fold increase in sensitivity to lometrexol compared with A431 cells in either low or physiological folate concentrations (Table 1) [31]. KB cells were f 20- and f 2-fold less sensitive to lometrexol in low (1 nM LV) and physiological folate (20 nM LV) medium, respectively, when 1 AM folic acid was added to competitively inhibit binding to the a-FR [31]. There has been some suggestion that the high affinity of lometrexol for the FR (particularly the hisoform) may be responsible for its uptake into liver [32]. However, the thiophene analogue of lometrexol, LY309887 was designed to be a poorer substrate for the FR. It was hoped that this modification would reduce uptake via the FR and reduce liver drug levels. In addition it is a better substrate for FPGS and inhibits GARFT more potently than lometrexol. However, in Phase I study delayed toxicity similar to that seen with lometrexol was observed, suggesting that FR-mediated uptake was not relevant to toxicity [32].

Raltitrexed, pemetrexed and ZD9331 bind to the RFC with an affinity similar to that of MTX (Table 1) and RFC-mediated transport is considered the primary mechanism of uptake into cells [7,31]. However, they also bind to the a-FR with high affinity. Pemetrexed (LY231514) has the highest affinity for L1210-FBP cell a-FR, ranging from 120% to 150% of folic acid using a competitive 3H folic acid binding assay [9,31] (Table 1). Raltitrexed and ZD9331 bind with slightly lower affinity ( f 60% of folic acid). Similar data were obtained using human A431-FBP cells [31]. All three drugs show a-FR-mediated activity in low folate conditions [9,31]. In physiological folate concentrations human A431-FBP cells are slightly more sensitive to these compounds compared with A431 cells (Table 1), or compared with A431-FBP cells coexposed to 1 AM folic acid to competitively inhibit binding to the a-FR [31]. KB cells displayed a similar difference in the presence of 1 AM folic acid except for raltitrexed that displayed no difference [31]. However, a major difference is apparent when cells are exposed to the agents for short time periods. For example, a 4 or 24 h exposure to ZD9331 in A431 cells (followed by removal of the drug containing medium and replenishment with drug-free medium (DFM) for the remainder of the assay time) gave IC50s of >100 and f 100 AM, respectively, whereas in A431-FBP cells the values were f 1.9 and 0.12 AM, respectively (Table 2) [31]. This has been ascribed to the fact that ZD9331 is not polyglutamated, and therefore, a-FR negative cells are relatively insensitive to short-exposure ZD9331 because of freely reversible transport. However, in a-FR overexpressing cells it is proposed that ZD9331 is trapped within the a-FR/endosomal apparatus leading to continuous delivery of ZD9331 into the cytosol [31]. Raltitrexed and pemetrexed are substrates for the intracellular enzyme, folylpolyglutamate synthetase (FPGS) and the high chain length polyglutamates formed are not readily effluxed from cells. Thus, short-exposure to these drugs leads to prolonged inhibition of TS in both a-FR positive and negative cells irrespective of any ‘‘endosomal trapping’’. In conclusion, antifolate drugs can bind to, and be transported by the a-FR, although RFC-mediated uptake is the predominant pathway. The clinical

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Table 2 Sensitivity of human A431 and A431-FBP cells to short-exposure raltitrexed, pemetrexed and ZD9331 Inhibition of cell growth IC50 (AM) A431

Raltitrexed Pemetrexed ZD9331

A431-FBP

4h

24 h

4h

24 h

0.054 F 0.017 15 F 2.6 >100

0.0059 F 0.0015 0.038 F 0.0060 >100

0.020 F 0.0006 2.9 F 0.35 1.9 F 0.50

0.0016 F 0.00006 0.012 F 0.0063 0.12 F 0.085

Cells (20 nM LV as folate source) were exposed to the drugs for 4 or 24 h and then washed and placed in DFM. Growth inhibition was measured using an MTT assay after f 4 cell doublings (72 h A431 and 96 A431-FBP). Data taken from Theti and Jackman [31].

relevance is difficult to assess but it seems likely that tumours with high overexpression of the a-FR may be more sensitive to these antifolates, particularly when exposed to ZD9331 for short periods of time. Therefore, it may be possible to assess aFR expression in individual patients and devise clinical protocols that exploit the predicted increased therapeutic index. More importantly, these studies provide a rationale for the design of antifolate drugs that are more selectively transported via the a-FR.

over-express the a-FR are sensitive to this agent, particularly when the extracellular folate concentration is low [9,34] (Table 3).

4. Targeting novel antifolate TS inhibitors to tumours via the A-FR Exploitation of differences in the biology of tumours and normal tissues is an important strategy for targeted therapy. One such difference is a-FR overexpression in some epithelial tumours such as ovarian, uterine and mesothelioma [1– 6]. Strategies include targeting imaging agents and antitumour agents to the tumour via folic acid- or a-FR antibody-conjugates (see other articles in this issue). Our own experience with designing antifolate drugs led us to discover compounds that are both the targeting agent and the cytotoxic agent without the requirement for conjugate synthesis. 4.1. Compound design The starting point for the design of a-FR targeted compounds was the polyglutamatable antifolate TS inhibitor CB3717 (Fig. 1) (reviewed in [33]). This polyglutamatable drug has very high affinity for the a-FR and relatively low affinity for the RFC (Table 3) [9,33]. Cell lines that highly

Fig. 1. Structures of quinazoline and cyclopenta[ g]quinazolinebased compounds.

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Table 3 In vitro properties of C2-methyl quinazoline and cyclopenta[ g]quinazoline derivatives

CB3717c ICI 198583 CB3900 CB300464 CB30523 CB30901 CB300638c

C2

C7

Ligand

Inhibition of L1210 TS, Kiapp (nM)

Inhibition of [3H]-MTX uptake, Ki (AM)a

Relative affinity for a-FR (% folic acid)b

NH2 CH3 CH3 CH3 CH3 CH3 CH3

H H CH3 C7 – C9 H CH3 C7 – C9

Glu Glu Glu Glu L-Glu-g-D-glu L-Glu-g-D-glu L-Glu-g-D-glu

20d 38 23 3e 4.6d 2.0f 0.33 F 0.16

46 F 17 2.6 F 0.058 4.3, 4.7d 9.1 F 5.1 94 F 27 269 F 162 393 F 231

150 F 12 80 F 5.2 30 F 6.6 44 F 10 72 F 8.7 31 F 0.89 66 F 8.3

Results are given as the mean F S.D. (n z 3) or as individual values. a Inhibition of 3H MTX uptake into L1210 cells [34]. b A whole cell [3H]-folic acid competitive binding assay using L1210-FBP cells [71]. c Theti et al. [34]. d Jackman et al. [72]. e Marriott et al. [43]. f Bavetsias et al. [37].

Phase I and II clinical studies were undertaken with CB3717 in the early 1980s (a-FR interactions were not known at the time) but clinical development was stopped due to dose-limiting nephrotoxicity that led to sporadic cases of serious toxicity when renal failure occurred in patients with myelosuppression [33,35]. Studies in mice identified low water-solubility of CB3717 in the acidic environment of the kidney tubules as the probable cause of the nephrotoxicity and led to the synthesis of C2-desamino-2-methyl compounds such as ICI 198583 [36], raltitrexed [14] and the non-polyglutamatable ZD9331 [17]. In addition, several series of non-polyglutamatable, quinazoline and cyclopenta[ g]quinazoline analogues were synthesised that were characterised by potent inhibition of isolated TS but low cytotoxicity against L1210 cells. Some of these compounds, particularly those with dipeptide ligands, displayed low affinity for the RFC [37,38] and thus were potential a-FR targeted agents. The compounds were characterised further by studying their relative affinities for the RFC and the a-FR along with their activity against a-FR expressing and negative tumour cell lines. Initial studies were carried out in the mouse L1210 leukemia cell line and its variants, the RFC negative L1210:1565, and the RFC negative, a-FR positive L1210-FBP line. The first two cell lines were cultured in commercial media containing supraphysiological concentrations of folic acid and the L1210-FBP cell line in low folate (2 nM

LV). To study the compounds in more physiologically relevant models, their activity was studied against human RFC positive A431 (a-FR negative), A431FBP (a-FR positive) and KB (a-FR positive) cell lines. These were performed generally in media containing a physiological concentration of folate (20 nM LV). These studies led to the identification of CB300638 as a lead compound with the required properties for an a-FR-targeted TS inhibitor [34]. Some of the findings of these structure – activity studies are summarised below. 4.1.1. Inhibition of isolated TS CB3717 and its more water soluble analogue, ICI 198583 inhibit isolated TS with Ki apps of 20 and 38 nM, respectively (Kis = 2 and 10 nM, respectively) (Table 3). The high chain length polyglutamates that can be formed inside cells are up to f 100-fold more potent inhibitors of TS [14,39]. The introduction of a C7-methyl function into ICI 198583 (to give CB3900) or replacement of the L-glu ligand of ICI 198583 with an L-glu-g-D-glu ligand (to give CB30523) increases TS inhibition f 2- and 8-fold, respectively (Table 3) [37,40,41]. Either modification prevents the compound being a substrate for FPGS [41,42]. Thus, the two compounds are important templates from which to develop a non-polyglutamatable a-FR-mediated TS inhibitor. Combination of both modifications gives the potent TS inhibitor, CB30901 with a Ki app of 2 nM [37]. Introduction of a C7 –C9 cyclopentane ring

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into CB3900 and CB30901 to give the cyclopenta[ g] quinazolines, 6S-CB300464 and 6S-CB300638, increases TS inhibition by an order of magnitude [34, 43]. CB300638 displays an affinity for TS that is at least 7-fold higher than the other compounds (Ki app = 0.33 nM) (Table 3). 4.1.2. Affinity for the folate transporters Compound affinity for the RFC was measured by their ability to inhibit [3H]-MTX transport in mouse L1210 cells. With the exception of CB3717, all of the compounds with a glutamate ligand displayed high affinities for the RFC with Ki values of 4 –10 AM (corresponding value for MTX is 3.2 AM) (Table 3) suggesting that the RFC is an important transporter for these compounds. In contrast, the dipeptide (L-glu-gD-glu) analogues had low affinity for the L1210 RFC with Ki values of >100 AM. A similar pattern of activity was observed in human W1L2 cells although the Ki values were f 2-fold lower [31]. Compound affinity for the a-FR (relative to folic acid) was measured in a competitive 3H folic acid binding assay in L1210-FBP cells. In this assay, CB3717 binds to L1210 a-FR with a higher affinity than folic acid (150%) but all the other compounds described here bind with lower affinity (30 –80% of folic acid) (Table 3). MTX has a low relative affinity of ( < 1%) in the same system. The relative binding affinities of CB3717, CB300464 and CB300638 were also measured using human A431-FBP cells and were found to be similar (data not shown and [34]). 4.1.3. Growth inhibition in mouse L1210 and L1210:1565 cell lines CB3717 is a relatively weak inhibitor of the growth of L1210 cells compared with its C2-methyl analogue, ICI 1985383 (Table 4). The f 30-fold increased potency of ICI 198583 has been ascribed to its increased efficiency for transport via the RFC and hence polyglutamate formation [36]. The non-polyglutamated compounds described in Table 4 have similar activities to ICI 198583 (IC50s f 0.2 AM) suggesting that their increased TS inhibitory activity is compensating for the lack of metabolism to more potent polyglutamate forms. However, CB300638 is 70-, 10-, 14- and 7-fold more potent as an inhibitor of isolated TS than CB3900, CB300464, CB30523 and CB30901, respectively, but all the compounds inhibit

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Table 4 Activity of CB3717 and C2-methyl quinazoline and cyclopenta[ g] quinazoline derivatives in mouse L1210 cell lines Inhibition of cell growth, IC50 (AM) L1210 (RFC+/FR CB3717 ICI 198583 CB3900 CB300464 CB30523 CB30901 CB300638

L1210:1565 )a (RFC /FR

5.0 F 1.2c 0.15 F 0.036c 0.21 F 0.036c 0.16 F 0.057 0.33 F 0.25c 0.22 F 0.22e 0.26 F 0.024

3.8 F 0.57c 5.8 F 2.7 8, 18 2.3 F 0.42 15, 16 8.0, 14e 2.1 F 0.31

L1210-FBP )a (RFC /FR++)b 0.0015 F 0.00062 0.00028d 0.0038d 0.0012 F 0.00035 0.004 F 0.002 0.0011 F 0.00076 0.00040 F 0.00018

a

L1210:1565 cells have a non-functional RFC. L1210 and L1210:1565 cells were cultured in standard RPMI 1640 medium that contains 2 AM folic acid as the folate source [42]. b L1210-FBP cells have a non-functional RFC and upregulated a-FR expression. Cells were cultured in folate-free RPMI 1640 medium (supplemented with 2 nM LV as the folate source) and dialysed foetal calf serum. c Jackman et al. [42]. d Westerhof et al. [9]. e Bavetsias et al. [37].

L1210 cell growth similarly (IC50s f 0.2 AM). This implies that CB300638 accumulates to a lower level in L1210 cells, at least in part because it is less efficiently transported via the RFC. The L1210:1565 cell line harbours a mutant form of the RFC and was less sensitive to all the compounds, suggesting that they may be transported into cells, at least in part, by the RFC. The potency of CB300638 for inhibition of isolated TS is 900-fold and 6000-fold higher than its inhibition of L1210 and L1210:1565 cell growth, respectively. Not only are these values higher than those of the other compounds in this series, they are very high compared with the RFC-mediated, nonpolyglutamatable TS inhibitor, ZD9331 (24- and 1400-fold, respectively). These results imply that CB300638 is transported with particularly low efficiency by the RFC in the L1210 cell line. 4.1.4. Growth inhibition in the L1210-FBP cell line The L1210-FBP cell line is an RFC negative mouse cell line with upregulated a-FR expression [44]. It is maintained by growing cells in medium containing very low levels of folate (2 nM LV). All the compounds in Table 4 were highly active in this cell line with IC50s of f 1 nM. These data demon-

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strate that the a-FR has the potential to be an alternative transporter for the compounds, at least in mouse L1210 cells grown in medium containing a low concentration of folate. 4.1.5. Growth inhibition in human A431, A431-FBP and KB cell lines A431-FBP cells are 5-fold more sensitive than A431 cells to CB3717 in 20 nM folate, suggesting that CB3717 is transported by the a-FR in this cell line (Table 5) [34]. However, in low folate A431FBP cells were 160-fold more sensitive than A431 cells [34]. Small differences between the two cells lines were observed for ICI 198583, CB3900, CB300464, CB30523 and CB30901 consistent with a low rate of a-FR-mediated uptake relative to other uptake mechanisms (Table 5). In contrast, the cyclopenta[ g]quinazoline, CB300638 was 300-fold more active against A431-FBP cells than A431 cells (Table 5) [34]. The IC50 is 0.0021 AM for A431FBP cells but increases to 0.97 AM in the presence of 1 AM folic acid added to the medium to competitively inhibit binding to the a-FR. Similar results were obtained in low folate medium [34]. KB cells are highly sensitive to the polyglutamatable compounds, CB3717 and ICI 198583 (Table 5). The fact that the activity of these compounds, particularly that of CB3717, is reduced in the presence of 1 AM folic acid suggests that a-FR-mediated uptake contributes to their activity. Of all the compounds in the series, the C7-methyl analogue, CB3900 displayed the lowest

potency in KB cells and its activity was the least affected by the co-addition of folic acid, suggesting that uptake by a-FR was not important in its activity. In comparison, the potent activity of the cyclopenta[ g]quinazoline analogue, CB300464 can be attributed largely to a-FR-mediated uptake as 1 AM folic acid increased the IC50 25-fold. The three dipeptide analogues (CB30523, CB30901 and CB300638) also displayed potent KB inhibitory activity that was reduced in the presence of folic acid (Table 5). CB300638 is the only compound in the series that displays potent a-FR-mediated activity in both A431-FBP and KB cell lines. These studies highlight the importance of the cyclopentane ring and the dipeptide ligand in reducing RFC-mediated uptake and possibly increasing the efficiency of a-FR-mediated endocytosis. CB300638 has been studied as a potential lead a-FR-targeted TS inhibitor and the findings of these studies are presented below. 4.2. CB300638 4.2.1. In vitro CB300638 possesses a number of in vitro properties important for an a-FR-targeted TS inhibitor. Its affinity for TS is very high with a Ki of 0.24 nM [34] and this is likely to be an important property if used to treat tumour cells in which a-FR-mediated trafficking may be slow. Even in the A431-FBP and KB tumour cell lines that express the a-FR at high levels, TS

Table 5 Activity of quinazoline and cyclopenta[ g]quinazoline derivatives in human A431, A431-FBP and KB cell lines

CB3717a ICI 198483 CB3900 CB300464 CB30523 CB30901 CB300638a

Inhibition of cell growth, IC50 (AM)

Inhibition of cell growth, IC50 (AM)

A431

A431-FBP

KB

KB + 1 AM FA

1.3 F 0.64 0.024 F 0.004 2.2 F 0.26 0.76 F 0.40 1.8 F 1.3 1.1 F 0.70 0.81 F 0.36

0.25 F 0.11b (5) 0.012 F 0.005 (2) 1.4 F 0.36 (2) 0.24 F 0.13 (3) 0.47 F 0.33 (4) 0.21 F 0.26 (5) 0.0028 F 0.0020 (290)

0.0067 F 0.0016 0.0021 F 0.00036 0.13 F 0.0058 0.0085 F 0.00042 0.0055 F 0.00076 0.010 F 0.0056 0.0036 F 0.0015

0.58 F 0.20c (87) 0.024 F 0.0024 (11) 0.31 F 0.036 (2) 0.21 F 0.0058 (25) 0.63 F 0.012 (110) 0.21 F 0.023 (21) 0.39 F 0.18 (110)

The A431-FBP cell line has been transfected with ha-FR and the A431 cells have been transfected with the empty vector. Cells were cultured in folate-free medium (supplemented with 20 nM-LV) and dialysed foetal calf serum [34]. IC50 values are given in the presence and absence of 1 AM folic acid. Growth inhibition was measured by an MTT assay after 72 h (A431) and 96 h (A431-FBP) and by cell counting after 72 h (KB). a Theti et al. [34]. b IC50 A431/IC50 A431-FBP. c Fold increase in IC50 in the presence of 1 AM folic acid.

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inhibition was induced slowly. The concentration of CB300638 that inhibited TS activity by 50% in intact a-FR negative A431 tumour cells decreased from 1 AM at 1 h to 0.11 AM at 16 h (Fig. 2) and was not affected by the co-addition of 1 AM folic acid [31]. This is consistent with uptake by mechanisms other than the a-FR, e.g. RFC. At 1 h, similar results were obtained in A431-FBP cells (IC50 = 0.79 AM) suggesting that a-FR-mediated uptake of CB300638 did not contribute to TS inhibition at this time. However, at later times the IC50 values for TS inhibition in A431FBP cells were lower than those obtained for A431 cells. For example, at 8 h the IC50 for A431-FBP cells was 0.0047 AM. KB cells displayed similar sensitivity to A431-FBP cells (Fig. 2). These data are consistent with the slow accumulation of CB300638 in a-FR positive cells to levels that inhibit TS and that are greater than levels achieved by transport through other mechanisms such as the RFC. Data discussed in Section 3.3 suggests that the aFR endosomal apparatus serves as a reservoir for ZD9331 leading to retention within and increased activity against a-FR positive cell lines under shortexposure conditions compared with a-FR negative cells. As might be expected this phenomenon is also observed with CB300638. A 1 h exposure of A431-

Fig. 2. Inhibition of TS in intact tumour cells. A431, A431-FBP and KB cells were incubated for the time indicated with increasing concentrations of CB300638 and the rate of 3H2O release from exogenously added [5-3H]-dUrd measured. The IC50 concentration was determined (concentration to inhibit TS by 50%). The experiments were conducted also in the presence of 1 AM folic acid to competitively inhibit CB300638 binding to the a-FR (this did not affect TS activity in A431 cells).

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Fig. 3. Inhibition of TS in intact A431-FBP and KB cells after 1 h exposure and 16 h washout. A431-FBP and KB cells were exposed for 1 h to 0.03 and 0.04 AM CB300638, respectively ( f 10  continuous exposure IC50s). Cells were washed and placed in compound-free medium for 16 h and then TS activity measured as described above.

FBP cells to 0.03 AM CB300638 did not lead to any TS inhibition at 1 h, but after the cells were washed and placed in DFM, TS inhibition increased with time [34]. For example, after 16 h in DFM, TS activity reduces to f 20% of control (Fig. 3). These data suggest that binding to the a-FR on the cell surface occurs within 1 h but that the trafficking and/or release of sufficient free drug to inhibit TS in the cytosol is a relatively slow process. The receptor recycling time of A431-FBP cells is not known but it is possible that the high number of receptors on the surface of these cells leads to sufficient delivery of CB300638 in one cycle. Therefore, cells expressing a lower number of receptors, or have slower recycling times may require longer exposure to CB300638 to induce a TS and growth inhibitory effect. Data obtained with KB cells (receptors f 50% of A431FBP) supports this. A 1 h exposure to 0.04 AM CB300638 followed by 16 h in DFM led to TS activity f 65% of control, i.e. less inhibition than was seen in A431-FBP cells after this time. These data may explain, at least in part, the lower activity of CB300638 in KB compared with A431-FBP shortexposure growth inhibition assays in which the endpoint for activity is measured after 72/96 h. The 1 h exposure IC50 for CB300638 in A431, A431-FBP and KB cells is >100, 0.8 and 18 AM, respectively. Near maximum activity was seen after 24 h (IC50 f 3

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AM), 8 h (IC50 f 0.003 AM) and 48 h (IC50 f 0.004 AM), respectively. 4.2.2. In vivo Pharmacokinetic studies were undertaken in mice bearing KB tumour xenografts. CB300638 was retained within the tumour at greater levels than in normal tissues [45,46]. Four hours after a 100 mg/kg

intraperitoneal (i.p.) injection, the normal tissue:plasma ratios were < 8 but the KB tumour:plasma ratios increased from f 20 to 100 after 16 – 72 h. In order to demonstrate that this localisation leads to selective TS inhibition in tumour compared with normal tissues, we conducted an in vivo pharmacodynamic experiment. TS inhibition was estimated using [125I]5-iodo2V-deoxyuridine ([125I]dUrd) metabolism [45,46]. IdUrd is a thymidine analogue whose uptake into tissues is proportional to the degree of TS inhibition [47,48]. Twenty-four hours after i.p. injection of 100 mg/kg of CB300638 or ZD9331, [125I]dUrd was injected and then 24 h after that, tissues and KB tumour were removed for g-counting. The results of one of these experiments are given in Fig. 4 and clearly demonstrate tumour-specific TS inhibition by CB300638.

5. Concluding remarks and future directions CB300638 is the first validated a-FR targeted TS inhibitor. It has the potential to have reduced normal tissue toxicity while maintaining antitumour activity in patients with a-FR expressing tumours. However, the experiments validating this approach have been performed using cell lines that express the a-FR to high levels. A number of questions have to be raised regarding how this approach will translate to the treatment of patients with a-FR-positive tumours. 5.1. a-FR expression levels

Fig. 4. The effect of ZD9331 and CB300638 on the biodistribution of IdUrd in mice bearing KB xenografts. Mice bearing KB xenografts were treated with 100 mg/kg CB300638 by i.p. injection. Twentyfour hours later they received 250 kBq [125I]IdUrd by iv injection then after another 24 h were killed and tissues removed for gcounting. Activities were normalised to tissue weight and expressed as the ratio of activity with tissue from mice treated with [125I]IdUrd but not CB300638 or ZD9331. Figures are mean F S.D. for three mice per group. * Indicates P < 0.05 compared with control values by one-way ANOVA with Bonferroni’s multiple comparisons test.

In order for a-FR-targeted TS inhibitors to be effective, the a-FR must be expressed at sufficiently high levels to transport enough drug to inhibit TS and cause cell death. It is important then to measure the expression of a-FR in both tumour and normal tissues. a-FR expression levels have been studied using different methods. Ross et al. measured the aFR mRNA levels in a number of human tumour cell lines and tumour samples from patients [2]. They concluded that a-FR mRNA expression in KB, JEG-3 and JAR-1 human tumour cell lines were similar but approximately an order of magnitude higher than levels in tumour types that expressed the a-FR to the highest level (ovarian and endometrial cancer). This suggests that human tumour cell lines expressing

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lower levels of the a-FR, e.g. the SKOV-3 may be more relevant in vitro models for evaluating a-FR targeted TS inhibitors. However, the relationship between a-FR mRNA level and protein expression is not completely understood. We have measured aFR expression in a panel of human tumour cell lines grown in 20 nM LV using a [3H]-folic acid binding assay or flow cytometry with the LK-26 monoclonal antibody. We found that the a-FR expression levels in both JEG-3 and JAR-1 were low relative to KB ( f 25% and 12%, respectively). Expression levels in the SKOV-3 were f 10% of KB (Green and Theti, unpublished data). Data to be published separately suggests that CB300638, and more particularly its 2hydroxymethyl analogue (CB300945) [46], inhibit the growth of JEG-3 and SKOV-3 grown in 20 nM LV by an a-FR-mediated mechanism (Green and Jackman, unpublished observations). However, selectivity for this mechanism is lost as the compound concentration increases. Although these studies suggest that the a-FR may be expressed to sufficient levels in tumours, it is not known whether the a-FR is functional. Further research is needed to determine whether tumour a-FR is more or less functional than that expressed in cell lines. It is possible that tumour function may be higher than cell lines. Tumour cell lines have been propagated for many generations in commercial medium containing supra-physiological concentrations of folic acid that can down regulate a-FR expression and the long term effect of this on function is unknown. 5.2. Evaluation of toxicity and antitumour activity in mice The toxicity of new drugs is usually evaluated in at least two species, including mice before testing in humans can begin. The antitumour and target (TS) related toxicities are difficult to evaluate in mice because rodents have plasma thymidine levels f100-fold higher than in humans [49,50]. This level can circumvent TS inhibition by providing an alternative source of thymidylate via the action of thymidine kinase [50]. Demonstrating the antitumour activity and toxicity of raltitrexed and ZD9331 in mice required that the drugs be given at high doses for long periods of time [51,52]. Studies have demonstrated that the chronic administration of TS inhib-

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itors such as CB3717 and raltitrexed to mice, lowers plasma thymidine, which may contribute to the activity of these drugs in mouse models ([50] and unpublished observations). The reduction in dThd may occur because of increased thymidine uptake by normal proliferating tissues following TS inhibition. This poses a significant problem for the evaluation of a-FR-targeted TS inhibitors in mice because they should not inhibit TS in normal proliferating tissues and thus should not deplete thymidine. This may mean that either we rely on pharmacodynamic endpoint measurements such as the thymidine analogue metabolism experiments, or conduct antitumour activity studies in mice with thymidine levels lowered using thymidine phosphorylase [53,54]. There are theoretical reasons to suspect that a-FRtargeted TS inhibitors might have non-TS related toxicities. a-FR expression in normal tissues is low but it is expressed to high levels in kidney and particularly on the brush border of proximal renal tubule epithelium [5,55,56]. The a-FR may have a role in reabsorption of folates (or antifolates) from urine back into the blood system and may explain, in part, the long terminal elimination phases of antifolate drugs. The nephrotoxicity of CB3717 in preclinical models and in clinical trials raises the possibility that part of its toxicity is mediated by a-FR in kidney. Although CB3717 binds tightly to the a-FR, it is more likely that the kidney toxicity of CB3717 is due to the low water-solubility in the acidic environment of the kidney tubules [33,57]. Although it is possible that aFR binding contributes to CB3717 kidney toxicity we believe that it is not a major contributing factor for this class of agent for the following reasons. Raltitrexed and ZD9331 both bind to the a-FR with similar affinity to CB300638 and are not nephrotoxic. In preclinical models high intravenous bolus doses of ZD9331 (200 mg/kg) reduced glomerular filtration rate and were associated with high kidney drug levels. Administering similar doses by i.p. injection did not affect renal function and was associated with 50-fold lower kidney drug levels [58]. The drug is given as a 1 h infusion in patients and does not cause kidney toxicity [52,59,60]. Kidney tubule epithelium does not proliferate rapidly and may be relatively insensitive to TS inhibition. Preliminary studies with CB300638 have shown that levels in the kidney are only 5-fold higher than

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in plasma 24 h after a single i.p. dose. This is a similar tissue-to-plasma ratio to that of ZD9331. In contrast, kidney raltitrexed levels are f 40-fold higher than plasma levels at the same time [61,62]. Mice have tolerated 100 mg/kg CB300945 (similar in vitro properties and pharmacokinetics) daily for 16 days with no weight loss or visible signs of toxicity suggesting that the toxicity profile of this class of compound is excellent (Forster and Jackman, unpublished data). 5.3. Clinical development It is worth considering the issues surrounding the clinical development of this class of a-FR targeted agent. First, the traditional approach to development of cytotoxic agents, e.g. determination of a maximum tolerated dose (MTD) and the use of the MTD in subsequent efficacy trials, is not relevant to the development of a targeted agent. More important is the determination of a biologically effective (TS inhibitory) dose in patients with a-FR overexpressing tumours, which theoretically should be lower than the MTD. Since the use of proliferating tissue toxicity as a biological surrogate marker of is not appropriate for a targeted agent, pharmacodynamic endpoints are needed to determine a biologically effective dose. We have successfully used increased plasma deoxyuridine (dUrd) as a surrogate marker of TS inhibition in clinical trials with ZD9331 [63,64]. However, the majority of this dUrd is likely coming from normal proliferating tissues rather than tumour. Therefore, it would be possible to use dUrd measurements differently in a Phase I clinical trial with an aFR targeted agent, i.e. to identify the minimum dose required to inhibit TS in normal tissues. This dose would then be predicted to be higher than required for optimal tumour selectivity. In parallel, it would be useful to measure biological endpoints in tumour tissues such as dUrd or TS expression (both rise upon TS inhibition) (unpublished data and [65]). It may be possible to use thymidine analogues with positron-emitting isotopes to show the localisation of TS inhibition by positron-emission tompography (PET) [66 – 68]. In a traditional Phase I trial, the toxicity and pharmacokinetics of a new agent are studied in patients with a range of different cancers. If a-FR-targeted

agents are assessed in this way, the MTD will be able to be assessed but not necessarily the effect on tumour TS in a-FR-expressing tumours. At some stage in the clinical development it will be necessary to stratify patients for a-FR expression, particularly if this class of agent is to be used in tumour types with a lower incidence of a-FR overexpression than ovarian cancer ( f 90% of ovarian cancer cases homogeneously overxpress the receptor). Methods for evaluating aFR expression may include immunohistochemistry on fresh frozen material [3] (the authors are not aware of the successful use in paraffin-embedded material) or measurement of RNA expression levels in paraffinembedded material (real-time PCR or in situ hybridisation) [4,6]. In ovarian patients it is possible to measure a-FR in tumour cells from ascites using flow cytometry or it may be possible to measure a-FR that is shed into plasma [69]. The effect of a-FR-targeted agents on a-FR expression needs to be considered. If a-FR expression is not related to critical events in carcinogenesis then tumour cells may be able to become resistant by downregulating a-FR expression or function. In addition, there is unlikely to be a substantial bystander effect, that is, the killing of cells within the tumour that do not express a-FR. For these reasons, a-FR-targeted agents may be best used in combination with other agents.

6. Conclusions In conclusion, a class of cyclopenta[ g]quinazooline-based TS inhibitors that are selective for a-FR expressing tumour cell lines in vitro are being tested in vivo. The first member of this new class of agent, CB300638 is highly selective for a-FR positive tumour cell lines in vitro and retains activity in media containing physiological folate concentrations. CB300638 is retained in a-FR-expressing KB xenografts following i.p. bolus administration. More importantly, thymidine analogue uptake studies have shown that this agent inhibits TS in a-FR-expressing KB xenografts but not in normal tissue. This class of agent needs further investigation but may result in a TS inhibitor that is active against a-FR expressing tumours and has reduced toxicity compared with conventional TS inhibitors.

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Acknowledgements This work is sponsored by Cancer Research UK and BTG. The authors would like to thank BTG for their very helpful comments during the production of this manuscript.

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