Cancer Treatment

Reversal

Keoiews [ 1990)

17 (Supplement

of multidrug

A), 37-43

resistance

S. B. Kaye CRC Department

qf

Medical

Oncology, lJniuersit_y of Glasgow,

1J.K.

Introduction The phenomenon of cross-resistance between certain specific cytotoxic agents was originally demonstrated experimentally 20 years ago, but its molecular basis has been elucidated only relatively recently (13). In the last few years, progrrss in understanding has been rapid, and interaction between basic scientists and clinicians has been clearly evident, so that clinical attempts to reverse this key process ofcytotoxic drug resistance are now underway. The basis for this so-called ‘multidrug resistance’ whereby tumour cells become resistant to natural products such as anthracyclines, podophyllotoxins and vinca-alkaloids, involves over-expression of the membrane glycoprotein -P-glycoprotein. This 170,000 dalton structure acts as an energy-dependent &flux pump, functioning to lower intracellular drug concentrations in resistant tumour ~11s. Present in certain normal cells, it is also present at increased levels in certain solid and haematological cancers, both prior to and following chemotherapy (9). Experimental studies indicate that reversal of the activity of this pump can be brought about by a range of membrane-active non-cytotoxic agents, most of which act by binding to P-glycoprotein themselves, thereby preventing cytotoxic drug efllux by competitive inhibition (25). In wishing to pursue these experimental observations into the clinic, certain problems need to be recogniscd. l!@cls on normal

tissues

High levels of P-glycoprotein are seen in normal organs such as the kidney, liver and gastrointestinal tracts. Histological studies indicate that it is disposed at the mucosal surfaces, suggesting that it subserves a normal excretory or protective role (22). Agrnts which aim to block P-glycoprotein function in tumour cells may therefore also lead to esccssive cytotoxic drug toxicity in normal tissues. Experimental studies have indicated that co-administration of the modulating agent, vrrapamil, does indeed lead to an increase in the gastrointestinal toxicity of vincristine in tumour bearing nude mice ( 11). There are also data to suggest that high doses ofverapamil increase the cardiac toxicity ofadriamycin in rabbits (2 1). However, there is no evidence that clinical use of modulators for reversing drug resistance has led to significant increases in toxicity in studies reported to date. It is

030.5 -7372/90/l

7.40037

+ 07 sjO3.00/0

I(‘# 1990 Axicmic 37

Prrss

Limited

38

S. B. KAYE

conceivable that relatively reversal of drug resistance

modest occurred.

increases

in toxicity

would

be justified

if significant

Interactions by dz$erence mechanisms jpharmacological) The range of agents which are known to reverse multidrug resistance experimentally includes verapamil. This is an agent with a variety of other actions, including effects on hepatic and renal blood flow. Clinical observations have confirmed that when verapamil is given along with adriamycin in an attempt to reverse drug resistance, a major factor is the impact on adriamycin pharmacokinetics, whereby its clearance is delayed and circulating levels are increased ( 14). This may have a more profound impact on the efficacy and/or toxicity of the cytotoxic drug than any effect acting via P-glycoprotein. Agents without these complicating properties would therefore seem to be more suitable modulating agents.

Ikgree ojprotein

binding

The majority of agents used clinically for attempts at modulation of multidrug resistance by P-glycoprotein binding are protein bound following administration. This means that extrapolation from in vitro to in vivo concentrations should be made cautiously, although it is evident that most in vitro studies have been carried out at relatively high (IO:&) serum concentrations, in which protein binding has presumably also occurred extensively. The degree of binding varies between 70 and 900,; for agents such as verapamil, quinidine, cyclosporin and amiodarone, all of which have undergone clinical testing. Recent in vitro data suggest that the impact of protein binding may vary from agent to agent, as judged by experiments in which the concentration of serum used in vitro has ranged from IOloo”,, (15). Cancer patients have increased levels ofal-acid glycoprotein, a serum protein component which is particularly involved in binding of basic modulating drugs. This factor therefore also needs to be taken into account (4)) as do the individual pharmacokinetic characteristics of the agents concerned. For example, a large volume of distribution would imply that tissue concentrations of the modulating agent may be much higher than those in plasma. Ideally concentrations at the tumour cell should be measured directly, and such studies are now underway.

kjicac~i ~J‘metabolites oJmodu1ator.r Many of the modulators which have been proposed for the reversal of multidrug resistance are metabolised extensively in vivo. Further investigations are required to determine whether these metabolites have a biological role as well. One example is verapamil, the major metabolite of which is norverapamil which is present in equimolar concentrations following its administration and which in fact does have comparable activity in vitro in reversing multidrug resistance ( 16). I n attempting correlations between in vitro and in uiuo concentrations of modulators, these metabolites require to be taken into consideration.

REVERSAL Table

1.

Correlation centrations

OF

MULTIDRUG between for MDR

in vitro modulators

Optimal in concentration

6 PM 6 PM

Verapamil D-vrrapamil Quinidinc Amiodaronc Bcpridil RO 1 l-2903

(DMDP)

‘l‘rifluopcraknr ‘1‘1-ans-nuprnthixol

RESISTANCE

vitro

and

plasma

39 con-

Clinically achievable plasma conrrntration I-2 PM ?

6 pw 2 /m 6 PM 1-4 PM I39 rig/ml 7

Cyrlosporin-A CtfOp-LWXlC ‘l‘amoxifcn ‘l‘orcmifctlc

Drug concentration/duration

of exposure

Extensive studies in vitro have determined the optimal concentrations of modulators for reversing multidrug resistance in tumour cell lines. For agents such as verapamil, quinidine and tamoxifen, concentrations of approximately 6 pM appear to be adequate for maximal reversal. Lower concentrations are effective to a lesser extent, although the majority of agents are ineffective at concentrations below 1 /lM. Most clinical studies have therefore concentrated on the use of modulators in which it has been possible to achieve circulating plasma concentrations in the range of l-6 PM (notwithstanding the considerations regarding protein binding outlined above). These correlations clearly take no account of potential differences in tissue rather than circulating plasma concentrations (see Table 1). It was quickly apparent in the early studies with the lead compound, verapamil, that clinical concentrations of 6 pm were very difficult to obtain without prohibitive cardiovascular toxicity (18). C oncentrations in the range of l-2 PM are easily achievable, but it may be that these are inadequate. Verapamil comprises a racemic mixture of two isomers in equal proportions, and in fact the n-isomer is rather less potent than the L-isomer in terms of its cardiac effects (7). Clinical trials are now in progress, aimed at assessing whether a significant advantage could accrue to the use of the o-isomer rather than the racemic mixture in terms of drug resistance reversal, since it is evident that n-verapamil possesses comparable modulatory activity in vilro (19). Experimental data have also shown that the duration of exposure of tumour cells to the modulating agent, such as verapamil, prior to exposure to the cytotoxic agent, does bear on the extent to which resistance is reversed (3). It seems likely that a minimum of 24 h of exposure is appropriate, and if the modulating agent is being given orally, some 2-3 days of treatment may be necessary to allow a plateau concentration to be achieved; this would depend on the half-life of the modulating agent concerned. I,evel oJ‘expre.r.rion of P-glycoprotein Histological studies have indicated that certain tumours such as renal cancer and colon cancer have high levels of P-glycoprotein. Presumably this reflects, to an extent, the high

40

S. B. KAYE

levels of P-glycoprotein in the organs from which these tumours arise. It would be logical to assume that clinical trials of multidrug resistance reversal would therefore most appropriately be carried out in these tumour types particularly as they are known to be among the most resistant of solid tumours. However, studies to date have been somewhat disappointing (see below), There are a relatively large number of other tumours in which P-glycoprotein is present in samples prior to therapy, but at lower levels than in renal and colon cancer. These include solid tumours such as breast cancer and ovarian cancer, and haematological cancers such as myeloma, acute myeloid leukaemia and non-Hodgkin’s lymphoma. When samples have been taken from patients with these tumour types following treatment, it has generally been noted that the level of expression of P-glycoprotein is higher (2). These tumours with a lower initial level of P-glycoprotein, may in fact also be appropriate for clinical trials of multidrug resistance reversal, particularly if the modulator is introduced at the beginning of therapy. Indeed, preliminary results from studies in haematological cancers are promising (see below). Co-esislence ofcellu1arfactor.s

governing drug resistance

The major interest in this particular form of drug resistance stems from the expanding information at the molecular level and the resultant tools which are provided for experimental work by the use of recombinant DNA technology. However, it should be recognized that there are several other mechanisms by which resistance to agents such as anthracyclines might develop. These include potential changes in levels of activity of the important nuclear target enzyme topoisomerase II (l), as well as increased levels of intracellular glutathione or of glutathione-s-transferase, both of which may act to increase detoxification of agents such as anthracyclines (5). It seems likely that co-existence of several factors together with increased expression of P-glycoprotein may occur in several clinical situations. Combinations of modulators may therefbre eventually be seen to be appropriate. Despite all these reservations clinical trials have proceeded in multidrug resistance reversal. 0~01s ~1 al. performed an initial small study with eight patients with rcfi-actory ovarian cancer, given a 24 h infusion of doxorubicin 50 mg/m2 together with a 72 h infusion of verapamil up to a maximum of 15 pm/kg/min. Although the median plasma levels of verapamil were in the range of 556 pm, an unacceptable level of cardiac toxicity was noted and the study was stopped ( 18). Despite this, other studies with verapamil have been performed. These include a smal! study by Presant el al. in which 13 patients received adriamycin 50 mg/m’ together with 480-960 mg of oral \.erapamil daily for 2 days. The maximum tolerated oral dose was 480 mg and in this study three ofeight patients refractor) to adriamycin had an objective response 120). Other non-randomized studies which involved oral verapamil have included a study from Figurredo ~1 al. of 58 patients with extensive stage small cell lung cancer treated with three drug chrmothcrapy plus oral verapamil up to 400 mg daily together with oral tamoxifen up to 100 mg daily fbr 3 days. They described a 58”,, overall response rate, with a median survival of 46 weeks, and suggested that these results may have been better than might have been achieved with chemotherapy alone (8). Randomized trials involving verapamil and both small cell and non-small cell lung cancer have been performed in three centers. Two of these, in Newcastle, UK, and in Sydney, Australia, are ongoing. The third, a large study iu Glasgow, UK, was completed

REVERSAL

OF

MULI‘IDRUG

RESIS’I‘ANCE

41

recently and comprised 220 patients with small cell lung cancer receiving 4-drug concentration chemotherapy with or without verapamil 480 mg daily for 3 days prior to chemotherapy. Mean concentrations of verapamil achieved were in the range of 1 pm. A recent analysis shows no overall difference in response rates (83% in the verapamil arm and 80%) on the chemotherapy alone arm) although there was a tendency to an increased complete response rate on the verapamil arm (38.5 vs. 270/,). However, no difference was seen in median survival between the two arms (41 vs. 44 weeks), although there was a tendency to an increased median survival for patients with extensive disease on the verapamil arm (32 vs. 22 weeks). No significant differences in toxocity were seen between the two arms, and overall it would seem that small cell lung cancer would not be an appropriate target for future studies given the information from more recent pathological studies suggesting that P-glycoprotein hyperexpression is very infrequently seen in this particular tumour type. More promising clinical studies with verapamil have been performed in a small number ofpatients with myeloma in pilot studies in both Arizona (6) and in London (IO). Patients with refractory myeloma have been retreated with chemotherapy together with oral verapamil in studies which would lead to concentrations ofapproximately 1 PM. Relatively short-lived responses have been seen in 20-30”,, of cases and these seem to correlate with the presence of P-glycoprotein in samples taken prior to therapy. These results have been considered encouraging enough to proceed with randomized studies in multiple myeloma, although it remains possible that the effect of verapamil in this situation may relate to changes in immunoglobulin synthesis through other mechanisms. Unconfirmed data also indicate that in patients with refractory non-Hodgkin’s lymphoma, a high level ofresponse is seen when chemotherapy is repeated together with continuous infusions of intravenous verapamil. A range of other modulators have been examined. These include a study by Miller el al., who treated 36 patients refractory to prior bolus dose doxorubicin with repeat doxorubicin, 60 mg/mY as a 90 h infusion together with trifluoperazine at a maximum tolerated dose of60 mg/day for 6 days. This led to a median plasma concentration of trifluoperazine of 130 rig/ml which was IO-fold less than the effective in r:itro concentration. Nevertheless, seven patients responded to treatment for a median duration of response for 4 months 117). Verweij et al. performed a clinical trial in 39 patients, 24 with colorectal and 15 with renal cancer, who were given chemotherapy together with cyclosporin A, 3 mg/kg as 2 1 h infusions 7 hours and 1 hour prior to chemotherapy. This comprised epirubicin 90 mg/m’ every 3 weeks for patients with colon cancer and vinblastine 0.1 mg/kg weekly for patients with renal cancer. The peak concentration of cyclosporin-A obtained was over 3000 rig/ml which is comparable to the effective in z&o concentration. Only one response was seen in one patient with colorectal cancer and the authors concluded that given in this form cyclosporin A was an ineffective modulator of multidrug resistance clinically

(23). In Glasgow, for multidrug P-glycoprotrin of28 primary with advanced given at doses concentrations

pathological studies have indicated that breast cancer may be a valid target resistance reversal. Using two monoclonal antibodies, positive staining for was seen in tumour cells as well as stromal cells in the majority of a series (untreated) samples (24). A pilot study was therefore performed in patients breast cancer treated with epirubicin 100 mg/m’ together with quinidine of 250 mg--- ~1 g twice daily. The aim was to assess whether poLentially active of quinidine could be achieved clinically without excessive toxicity. This

S. B. KAYE

42

study of31 patients confirmed that this was indeed the case (Jones e/ al., 1989). Although side-effects were seen at the higher doses, at 250 mg b.d. the side-effects were minor and the mean concentration achieved was 6 PM, which was within the effective in vitro range. No sign ofincreased toxicity ofepirubicin was seen, and for this reason a placebo-controlled double-blind trial is now underway using quinidine for 4 days prior to treatment with epirubicin for advanced breast cancer. Finallv. another agent tested as a modulator has been tamoxifen, even though it may be that its effect in reversal of multidrug resistance is through a diRerent mechanism. A Phase l/II trial in Newcastle, UK, has used oral etoposide 300 mg daily for 3 days together with oral tamoxifen up to 480 mg per day for up to 6 days. The projected concentration of tamoxifen is in the range of 4-6 ,MM which approximates to the effective in vitro concentration. This dose appears to be achievable without significant clinical side-eflects. Six objective responses were seen in the first 46 cases treated in this study (2). As is evident, most of the agents for multi-drug resistance reversal have been selected empirically and were not synthesised for this purpose. As information on the characteristics of binding to P-glycoprotein emerge, agents specifically synthesised for this purpose are being developed. More efficient inhibition of P-glycoprotein function without unwanted side-eft‘ects, may therefore be anticipated. It may therefore soon be possible to determine, more accurately, the impact which attempts at multidrug resistance reversal will have on clinical practice, and it is the author’s prejudice that at least in haematological cancer, that impact will be considerable.

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It’.

‘I‘. (1989)

Unknotting

the complrsitics

of multidrug

rcristance:

the involvement

of DNA

topo-

isomrrasea in drug a&on and resistancr. ,/, XCZ//. Cancer In,% 81: 1683-1685. 2. Cantwell, B., Carmichael, J., Millward, M. J,, Chattcrjer, M. & Harris, A. L. (1989) Intermittent high dose tamuxifcu (HD’I‘) with oral ctoposide (E1’0): Phase I and II clinical studies. HP-. ./. Cnncrr 60: 450. 3. C&s. C. E., Janowska-~Ti~rzorek, Elf&-t ofduration ofexposurc Cmcer I&J. 49: 5798-5804. 4. Chattcrjer, modulation

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6. Dalton, IV. S.. Grogan, ‘I‘. .Ll., ,\lrltzu. P. S., Srhrpcr. R. J,, Duric, B. M., ‘l‘aylor, C. \I’.. Miller, & Salmon, S. E. ( 1989) Drug rcsistanw in my&ma and lymphoma: detection of P-glycoprotrin potential circumvrntion I))- addition ofwrapamil. J. C’lin. O/rco/. 7: 415.-424. 7. Echizcn. on A-\’

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‘l‘hc r1fcc.t ofd.

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vcrapamil

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REVERSAL

lation

by

xcnografts.

vcrapamil

of

vincristine

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RESISTANCE

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and

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mice

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.Llvrr>, S., E‘lanigan, P., Schlick. E:.. Frcshney, R. 1. Br Kayc, S. B. (1989) in a murine tumour line: circumvention with verapamil and norverapamil. 17. Miller, R. L., Bukowski, R., Budd, (;. ‘I’., l’urvis, J., \Vcick. J. K., Shcphard, ( 1988) CXnical 6: 880 -888.

18. Cb.ols, (1987)

modulation

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S. B. i 1990) ‘l‘hr

activity

human tumour ~11 lines ia not srcrtwspccific. C;. A., Kcnncdy, I’. S., b’iwman, C., Gala. rcvcrsal of clinical doxoruhicin rcsistancc

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localization

ofthc

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rcuistancr

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of verapamil

as a resistance

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of multidrug

wrzapamil to P-glycoprotcin on spccilic sites, and transport Ixanc of K562/AD.\i ~11s. GTM(Y~ Re.wnrrA 49: 5002-5006.

rrsistance

of verapamil

T. Pr Kayc, S. B. (1990) with two monoclonal

by vcrapamil: outward

across

direct

binding

the plasma

mem-

of