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21 Trials and Tribulations in Developing Clinical Trials of Gene Therapy

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Summary

At The University of Texas M.D. Anderson Cancer Center, we have been investigating use of the adenovirus type 5 E1A gene as a cancer therapy for breast or ovarian cancer since 1995. E1A gene therapy is a nonimmunologic approach involving ectopic expression of a potentially therapeutic gene in cancer cells. During the late 1980s, E1A was shown to downregulate the overexpression of HER-2/neu, thus reversing the tumorigenic and metastatic phenotype of HER-2/neu-overexpressing breast and ovarian cancer cells. Since that time, E1A has also been shown to function as a tumor suppressor gene in several other types of tumor cells by inducing apoptosis and differentiation and by inhibiting metastasis regardless of HER2 expression level. This chapter highlights some of the issues and difficulties we encountered in designing clinical trials of E1A gene therapy for breast or ovarian cancer.

Key Words: Cationic liposome; breast cancer; ovarian cancer; adenovirus type 5 E1A; clinical trial.

From: Cancer Drug Discovery and Development: Gene Therapy for Cancer Edited by: K. K. Hunt, S. A. Vorburger, and S. G. Swisher © Humana Press Inc., Totowa, NJ

387

21 Clinical Trials of Gene Therapy

E1A for Breast or Ovarian Cancer

Naoto T. Ueno,

MD

,

PhD

, Judith K. Wolf,

MD

, David M. Gershenson,

MD

,

Gabriel N. Hortobagyi,

MD

, and Mien-Chie Hung,

PhD

C

ONTENTS

ADENOVIRUSTYPE5 E1A: ANONCOGENE AND ATUMORSUPPRESSOR GENE

SELECTING AGENEDELIVERYSYSTEM PRECLINICALSTUDIES

THEPHASEI TRIAL

TRIBULATIONS INDESIGNINGCLINICALTRIALS PHASEII STUDY OFE1A MONOTHERAPY RATIONALE FOR ANEW ANDIMPROVEDTRIAL

NEWPHASEI/II RANDOMIZEDTRIAL OFE1A GENETHERAPY WITH

PACLITAXEL FINALTHOUGHTS

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1. ADENOVIRUS TYPE 5 E1A: AN ONCOGENE AND A TUMOR SUPPRESSOR GENE

Within the adenovirus genome, the E1A gene encodes proline-rich nuclear phospho- proteins that regulate the efficient replication of the adenovirus (1). Because E1A proteins can transcriptionally transactivate proteins that can “immortalize” transformed cells, E1A was originally considered an “immortalization oncogene” (2–4). Certain serotypes of E1A proteins, such as those of adenovirus type 12, are strongly oncogenic and induce tumors at high frequency (1). Others, such as adenovirus type 5 and its closely related serotype adenovirus type 2, are nononcogenic (5). We found that por- tions of E1A gene products inhibited HER2 overexpression in both rodent fibroblasts and human cancer cells, and they did so through transcriptional repression at the HER2 promoter (6,7). This effect was thought to result from stimulation of transcription or repression of the activity of viral and cellular transcriptional enhancers.

These findings prompted us to investigate the possibility that E1A might function as a tumor suppressor gene in HER2-overexpressing cancer cells through repressing HER2 overexpression. First, to determine whether E1A could reverse the tumorigenicity of HER2-overexpressing human ovarian cancer cells, we transfected several such cell lines with the E1A gene and found that the transfected cells expressed less HER2 protein, produced fewer malignancies in immunosuppressed animal models, and were less able to induce tumors in immunocompetent mice (8).

In addition to this tumor-suppressive activity in HER2-overexpressing cells, E1A has other suppressive activities independent of HER2 (9,10). E1A has been shown to repress the transcription of various proteases involved in tumor cell invasion and metastasis, including type IV collagenase (11,12), plasminogen activator (13), stromelysin (14), interstitial collagenase (11), and urokinase (11); E1A can also inhibit metastasis by elevating expression of the metastasis suppressor gene Nm23 (15,16). E1A can also reduce the anchorage-independent growth and tumorigenesis of a variety of tumor cell lines, including human melanoma, fibrosarcoma (17), rhabdomyosarcoma, Saos-2 osteosarcoma, non-small cell lung carcinoma, breast carcinoma (18), and murine melanoma (19), either by controlling cell proliferation through repression of growth factor-inducible genes (14) or by inducing differentiation (20). E1A can also induce apoptosis in various cell types (19,21–24). E1A was recently shown to suppress at least one other tyrosine kinase besides HER2—namely Axl, the prototype of a family of transmembrane receptors that also includes Sky and Eyk (23,25), which may be involved in apoptosis or suppression of cell proliferation. E1A can also induce immune responses by sensitizing cells to apoptosis induced by tumor necrosis factor (TNF)-F (26,27) and cytolysis by activated natural killer (NK) cells and macrophages (26,28,29).

In summary, E1A functions as a tumor suppressor gene through several mechanisms (9,10): transcriptional repression of HER2, inhibition of metastasis-related genes, acti- vation of metastasis suppressor genes, induction of differentiation or suppression of cell proliferation, induction of apoptosis, and induction of host immune responses.

2. SELECTING A GENE DELIVERY SYSTEM

Armed with these results, we then set out to choose a gene delivery system for our preclinical and clinical trials. Human gene delivery systems are of two types—viral or nonviral. Viral delivery of DNA plasmids is efficient, but the disadvantages include the inability of retroviral vectors to transfect nondividing cells and the strong immunogenicity

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of some adenoviral vectors (30). Our initial choice for our preclinical animal studies was to use a replication-deficient adenoviral vector (31) to avoid potential problems with low-transfection efficiency. On the other hand, we reasoned that nonviral delivery systems might be more appropriate for clinical studies because they could be used repeatedly with minimal toxicity and immunogenicity; however, the transfection efficiency of such systems tends to be more limited than that of viral vectors (32). One of the best-known nonviral delivery systems at that time was the cationic liposome, the first of which was developed for in vitro gene transfer in the late 1980s by Felgner and others (33). The system was designed so that the overall charge remains positive after formation of the DNA/cationic liposome complex; this positive charge would promote the interaction of the complex with the negatively charged cell membranes and enhance transfection of the target cells. For our studies, we chose the 3-[N-(Ne,Ne-dimethyl- aminoethane)carbamoyl] cholesterol (DC-Chol) cationic liposome developed by Leaf and colleagues at the University of Pittsburgh. This system was shown to facilitate gene delivery into mammalian cells both in vitro and in vivo and was relatively nontoxic as well as being biodegradable, nonmutagenic, and nonimmunogenic (34–38).

3. PRECLINICAL STUDIES

3.1. Safety and Efficacy of E1A in Xenograft Mouse Models

The first steps in the development of E1A as a therapeutic agent for human cancer involved establishing the antitumorigenic efficacy of E1A by using adenoviruses to trans- fect HER2-overexpressing ovarian or breast cancer cells and examining the behavior of those cells in vitro and upon their injection in mice (31). The next steps involved using E1A, delivered by various means, to treat breast cancer or ovarian cancer in xenograft mouse models. In one such study, nude mice were injected in the mammary fat pads with the human HER2-overexpressing breast cancer cell line MDA-MB-361 and tumors were allowed to form. Then E1A genes complexed with DC-Chol cationic liposomes (E1A/DC- Chol complex) were injected into the tumors (39),where they were shown to inhibit the growth of these breast cancer cells. Similar results were observed in an intraperitoneal (ip) model of ovarian cancer involving the HER2-overexpressing cell line SKOV-3; in that study, ip delivery of the E1A/DC-Chol complex inhibited the growth and dissemination of these ovarian cancer cells. About 70% of these mice survived for at least 365 d, whereas all untreated controls developed severe tumor-related symptoms and died within 160 d (38).

3.2. Toxicity Studies

The final step before the phase I trial was to evaluate the toxicity (safety profile) of the E1A/DC-Chol complex after ip injection in normal mice. In the short term, cumula- tive doses of up to 40× those proposed for the phase I trial showed no adverse effects on renal, hepatic, or hematologic function in nude mice, nor were any major pathologic changes observed in any organs at necropsy. At 9 mo after discontinuation of treatment, no macroscopic or microscopic effects were noted in the genitals (uterus, fallopian tube, and ovary), liver, lung, heart, kidney, spleen, or brain. At 18 mo after treatment cessation, E1A DNA could still be detected in lung and kidney but not in liver, heart, spleen, brain, uterus, or ovary. Our conclusions from these tests were that the DC-Chol cationic liposome gene delivery system could allow repeated dosing of the E1A/DC- Chol complex without inducing any major toxicity but that long-term follow-up was needed to determine the ultimate effects of E1A (36,37).

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4. THE PHASE I TRIAL

On the basis of these results, an Investigational New Drug application was filed with the United States Food and Drug Administration and approved, and a phase I clinical trial of E1A gene therapy for metastatic breast or ovarian cancer was opened at MD Anderson Cancer Center in 1996 (40) (see Fig. 1). The goal of this study was to demon- strate the feasibility of the therapy and its effectiveness as assessed by downregulation of HER2 overexpression in tumor tissue. At first, only patients with tumors that over- expressed HER2 were eligible; the eligibility criteria were later expanded to include patients with tumors expressing low levels of HER2. Each patient was to receive a weekly injection of the E1A/DC-Chol cationic liposome complex through an indwelling Tenckhoff catheter placed in either the pleural cavity (for patients with breast cancer) or the peritoneal cavity (for patients with ovarian cancer). Each cycle consisted of three consecutive weekly injections followed by 1 wk off. The starting dose for the E1A/DC- Chol complex, 1.8 mg/m2/injection, was derived from the effective dose established in the preclinical animal studies (38,39). Three dose levels were tested: 1.8, 3.6, or 7.2 mg/m2/injection.

4.1. Outcome

The median number of E1A/DC-Chol complex injections was 6 (range 1–8); the most common adverse event was development of fever 2 to 3 d after each injection regardless of dose. The appearance of grade 3–4 nausea/vomiting or pain in the 7.2 mg/m2dose group led us to reduce the MTD to 3.6 mg/m2. Three patients experi- enced catheter-related infections requiring discontinuation of the treatment. No drug- related mortality was experienced. Several patients experienced stable disease with transient decreases in levels of the tumor markers CA-125 or carcinoembryonic anti- gen. Immunohistochemical staining of tumor tissue for HER2 expression showed downregulation of HER2 in all 6 patients so tested. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of tissues from one patient at autopsy revealed E1A expression in lung, tumor, liver, and kidney but not in brain or ovary. E1A expression in tumor tissues was also confirmed by immunohistochemical staining, as was induction of apoptosis, after treatment with the E1A/DC-Chol complex. We concluded from these results that E1A delivery is feasible and that E1A expression could be detected in can- cer cells after such treatment.

5. TRIBULATIONS IN DESIGNING CLINICAL TRIALS 5.1. Monitoring Gene Expression: Choice of End Point

One of the most difficult challenges in clinical gene therapy is planning how to moni- tor the presence and effects of the delivered therapeutic gene in vivo. The end points selected for our phase I trial were the maximum tolerated dose (MTD) and the maxi- mum biologically active dose (MBAD) of the E1A plasmid. Because no toxicity had been detected during the preclinical toxicity study, we initially thought that we might not reach the MTD, and thus the dose of E1A plasmid was escalated in 100% incre- ments rather than by the Fibonacci method. MBAD, the primary end point of the study, was defined as the dose of E1A plasmid (in mg/m2) that would produce at least a 25%

downregulation in HER2 expression in the tumor—the best-established end point at the time the trial was designed.

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5.2. Choice of Measurement Techniques

Theoretically, the expression of a therapeutic gene in both targeted and nontargeted cells can be monitored at three levels: at delivery of the plasmid DNA into the cells (transfection), at mRNA synthesis from DNA (transcription), and at protein synthesis from mRNA (translation). Assays are available for monitoring gene expression at each of these levels (Table 1).

When designing a clinical trial, assay selection is critical for several reasons. First, the assays can be expensive and time-consuming. Second, the choice of assay is often affected by the type, source, and volume of tumor (or nontarget) tissue that can be col- lected for analysis. In our phase I trial, we chose immunohistochemical staining, which would reveal both the presence and the distribution of the E1A and HER2 protein pro- ducts in tissue, and RT-PCR assays to detect E1A RNA. Western blotting was not consi- dered feasible because it requires large amounts of E1A-transfected tissues.

5.3. Other Considerations: Efficiency, Distribution, and Duration of Gene Expression

Other issues to be considered before initiating our phase I trial were how to assess the efficiency, distribution, and duration of expression of the therapeutic gene. The optimal method for accurately determining the transfection efficiency of therapeutic genes in both tumor and nontumor cells in human subjects has yet to be established. In preclinical models, the efficiency of DNA transfection or protein expression is com- monly described as a percentage of cells that stain positively for gene or protein expres- sion among the entire cell population exposed to the transfecting agent. In intraperitoneal or intrapleural therapy, transfection of the E1A/DC-Chol complex into human cells is probably limited to those cells that were in direct proximity to or in con- tact with the complex, making measurement of gene expression difficult. Similar findings

Fig. 1. Study concept of the phase I trial of E1A gene therapy in breast and ovarian cancer.

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have been reported from intratumoral gene therapy trials: expression of the gene tends to be strongest at the point at which the needle was inserted into the tumor.

Monitoring the distribution of therapeutic genes, even in local gene delivery sys- tems, requires examining gene distribution in a variety of organ sites. We attempted to address this issue in our E1A/DC-Chol trial by collecting tissue samples from as many organs as possible at autopsy. This requirement was particularly acute because cationic liposomes can be distributed systemically even after local delivery. As for the duration of effect, many questions remain unanswered as to the transience or stability of gene expression in cancer gene therapy. Expression of genes delivered by cationic liposomes is transient in cells, and the DNA plasmid is degraded relatively quickly. However, we do not know how long the gene may be expressed in the cells, nor whether (or when) the transfected gene might become integrated into the host chromosomes. The potential for integration is a critical safety concern in gene therapy, as the integrated DNA could confer chronic adverse effects to normal host cells or even be passed on to the next generation if the transfected cells reside in the organs of fertility (e.g., the ovaries).

Issues such as these are even more important for systemic (as opposed to local) gene delivery systems.

5.4. Accessibility of Tumor Tissue Samples

Another topic addressed in the design of this clinical study was the need to collect serial tumor specimens over time. Because intracavitary fluids are likely to contain tumor cells, we initially included only patients who had either ascites or pleural effu- sions, reasoning that insertion of a Tenckhoff (indwelling) catheter would allow repeated (weekly) fluid samples to be collected readily, with less risk and discomfort to the patient than other more invasive means of sampling tumors. We also considered that this approach might allow us to determine the ideal sampling intervals in terms of maximal transfec- tion and protein synthesis after injection of the E1A/DC-Chol complex. Thus, our plan was to collect specimens before the first injection of E1A/DC-Chol complex and again 3 and 7 d thereafter. However, the wisdom of this approach remains controversial with regard to how aggressively one should attempt to collect tumor samples in light of the expense and the difficulty in obtaining samples in many clinical trials.

5.5. Monitoring Biological Effects

When we began designing the phase I study in 1995, we attempted to select an MBAD that would serve as the basis for establishing dosages for future phase II trials.

Our reasoning was based on the assumption that the molecular effect (HER2 down- regulation) triggered by the therapeutic gene (E1A) would be the dominant mechanism of antitumor activity. Our thought was that E1A may downregulate HER2 at dose A,

Table 1

Assays for Monitoring Gene Expression

DNA RNA Protein

Southern blotting Northern blotting Immunohistochemical staining Polymerase chain reaction Reverse-transcriptase Western blotting

polymerase chain reaction Fluorescence in situ

hybridization

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but to induce rapid apoptosis, it may have to be given at a higher or lower dose.

Alternatively, low E1A gene expression may induce strong antitumor activity by trig- gering a bystander effect, which could depend on the dose of the therapeutic gene or on a particular genetic abnormality in the cancer cells.

However, in the end, we abandoned the attempt to use MBAD to define the dosages for the phase II trial. Despite the confirmation that E1A had a biological effect at a dose lower than the MTD, the traditional MTD was used instead, for the following reasons.

First, the possible therapeutic mechanisms suggested by the preclinical experiments do not always match the antitumor mechanism in clinical settings. Research by us and others suggested that E1A could reverse the malignant phenotype through more than one mechanism,(e.g., by triggering the host immune system), inducing apoptosis, inducing tumor lysis, and suppressing metastatic capability (41). Even if antitumor activity could be detected in the treated patients, other activities associated with the E1A/DC-Chol complexes could have contributed to the antitumor effect in addition to the down- regulation of HER2. In attempts to clarify these issues, we studied several indices in several types of samples (i.e., tumor, intracavitary fluid, and serum) in the phase I trial. For example, we studied apoptosis and Ki-67 expression (an index of cellular proliferation) because E1A is known to induce apoptosis and suppress proliferation of certain cell types; we also measured cytokines (TNF-F and interferon [IFN]-L) in the cavitary fluid because E1A is suspected of sensitizing cells to TNF-F.

Another problem was that assays to measure apoptosis, Ki-67, and cytokines had not been validated with clinical samples, and so any retrospective analyses of samples col- lected prospectively from phase I participants were limited by the possibility that the samples may not have been collected under the appropriate conditions or at appropriate times. Hence, the appearance of apparent associations among variables generated did not confirm the molecular mechanism of E1A; rather, they generated new hypotheses that needed to be tested in future trials.

By this reasoning, an accurate and useful MBAD for use in phase II trials would be difficult—if not impossible—to choose. In gene therapy trials for cancer, the threshold for triggering the molecular effect of a therapeutic gene is generally not known. This does not mean that one should not make an effort to determine these factors. Rather, a tremendous effort is needed to identify biological markers and at the same time come up with assays that can allow those markers to be monitored in a scientifically valid and reproducible manner.

6. PHASE II STUDY OF E1A MONOTHERAPY

As discussed above, the findings from our phase I trial actually led to additional hypotheses to test in both preclinical and clinical experiments. Our initial phase II study, which was to open in 2000, was designed to address some of these hypotheses, especially transfection efficiency. With this in mind, we restricted the study criteria to include only patients with ovarian cancer with relatively small tumors (i.e., 2 cm or less in largest diameter) and hence a generally better prognosis that those with metastatic disease. However, this restriction led to major difficulties in patient accrual, because several other potentially more attractive treatment options were open to such patients. In addition to the difficulties in competing for a relatively small pool of patients to participate in several clinical trials, encouraging patients to participate in gene therapy trials was particularly difficult after an 18-yr-old patient

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with an inherited enzyme deficiency died while participating in a clinical trial of human gene therapy in 1999. These difficulties led to premature closure of our phase II monotherapy study in 2002.

7. RATIONALE FOR A NEW AND IMPROVED TRIAL

After our experiences with the preclinical, phase I, and phase II studies recounted above, our next step was to open a phase II trial of E1A gene therapy used in combina- tion with paclitaxel for ovarian cancer, with the hope that the addition of E1A would enhance the antitumor activity of paclitaxel and improve long-term outcome. Our ini- tial hypothesis was that the ability of E1A to downregulate HER2 in HER2-over- expressing breast cancer cells could sensitize those cells to paclitaxel. Moreover, we showed that a paclitaxel-resistant phenotype in breast cancer cells was associated with enhanced p185 expression and that downregulation of p185 by E1A converted this resistant phenotype into a sensitive one (42). These in vitro data led us to conduct a study in which E1A chemosensitization was tested in xenograft models of human HER2-overexpressing breast and ovarian cancer in which E1A gene therapy was com- bined with paclitaxel.

As noted earlier in this chapter, a major problem with gene therapy that remains to be resolved is how to deliver the therapeutic gene to distant target tissues. In our phase I trial of E1A gene therapy, delivery of the E1A gene into either the thoracic or the peri- toneal cavity by means of DC-Chol cationic liposomes led to successful transfection of cancer cells. Use of the DC-Chol system is limited to intratumoral or intracavitary regional delivery, however, because of the vulnerability of the DNA/DC-Chol complex to serum. To overcome this limitation, another liposomal delivery system, the lipid- protamine-DNA or LPD system, was developed. In this formulation, the DNA is con- densed, as it would be in natural vectors such as viruses, in a manner that makes it more resistant to degradation by serum. Injection of LPD complexes via the tail vein in nude mice has been shown to facilitate the delivery of the DNA to distant organ sites such as lung or liver (43–45).

We used a version of this LPD system, LPDI, to deliver E1A gene therapy in combi- nation with paclitaxel to mice implanted with human breast or ovarian cancer cells that overexpress HER2. Mice in the control groups (i.e., those given only E1A plasmid, only liposome, or a complex with a nonspecific gene) showed no suppression of tumor growth (46,47). Mice treated with either the E1A/LPDI or paclitaxel showed some evi- dence of tumor suppression, but mice treated with the E1A/LPDI-plus-paclitaxel com- bination showed remarkable suppression of tumor growth. These findings led us to conclude that the E1A gene can enhance the sensitivity of HER2-overexpressing human breast cancer cells to paclitaxel and that intravenous (iv) delivery of E1A for gene therapy is in fact a feasible approach.

Another line of evidence indicates that the sensitization conferred by E1A is not lim- ited to paclitaxel or to cancer cell lines that overexpress HER2. One group reported that p53-dependent apoptosis induced by E1A sensitized cells to the alkylating agent cis- platin (21). Other groups have reported sensitization to cisplatin via a p53-independent mechanism (48–50). Still another group has reported sensitization to cisplatin, etopo- side, and gemcitabine by E1A in low-HER2-expressing cancer cell lines (18,51,52).

This chemosensitization by E1A was further confirmed in a xenograft model with the low-HER2-expressing breast cancer cell line MDA-MB-231 (53).

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8. NEW PHASE I/II RANDOMIZED TRIAL OF E1A GENE THERAPY WITH PACLITAXEL

On the basis of the findings described above, we are now planning a phase I/II trial targeting platinum-refractory ovarian cancer (see Fig. 2). The purpose of this random- ized trial is to compare weekly paclitaxel vs weekly paclitaxel plus E1A gene therapy.

The primary objectives are to evaluate the toxicity and establish the MTD of ip E1A/DC- Chol complex in combination with iv paclitaxel and to compare tumor response between treatment groups. Secondary objectives are to compare time with progression and over- all survival and to examine the biological effects of the E1A/DC-Chol complex + pacli- taxel combination on ovarian cancer cells through laboratory testing.

The study was designed to address whether the addition of E1A gene therapy is supe- rior to weekly doses of paclitaxel, which is known to have antitumor activity in plat- inum-refractory disease. Our hope is that having attractive treatment options for both groups, and no limitations on tumor size, will improve accrual to the study.

Our plans for testing biological activity, derived in part from our phase I results, include measurements of apoptosis, HER2 downregulation, Ki-67, and cytokines (e.g., TNF-F) in ascites. This attempt to monitor potential biomarkers in a prospective man- ner is important for maintaining ongoing, bidirectional links between clinic and labora- tory. Approaches that involve prospective study of molecular events that can trigger antitumor activity or side effects are crucial for improving the efficacy and safety of cancer gene therapy.

9. FINAL THOUGHTS

As is true for any other type of study, simply performing a clinical trial of cancer gene therapy is no guarantee of future success. Many attractive new therapeutic modal- ities that compete with gene therapy are available to patients. Mechanism-oriented monitoring strategies are needed that may allow the identification of new response pre- diction markers or improvements in the efficacy of therapeutic genes during the course

Fig. 2. Study design of the phase I/II trial of E1A gene therapy and paclitaxel.

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of the trial. Results from phase I trials revealing some of the tumor suppression activi- ties associated with E1A have already gone some way toward establishing this flexibil- ity by laying the scientific groundwork for phase II clinical trials of E1A as an anticancer agent. In the end, however, any study, in addition to being ethical, must offer an attractive range of potential treatment options if it is to appeal to patients who might be persuaded to participate.

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