Bowman Birk Inhibitor Concentrate and Oral Leukoplakia: A Randomized Phase IIb Trial

Abstract

Oral premalignancy serves as an ideal model for study of chemopreventive agents. Although 13-cis-retinoic acid demonstrated reversal of oral premalignancy, toxicity and reversal of clinical response after cessation of therapy obviated its widespread use. A search for nontoxic agents with cancer preventive activity led us to evaluate Bowman Birk Inhibitor (BBI) formulated as BBI Concentrate (BBIC). We previously reported encouraging results in a phase IIa trial of BBIC in patients with oral leukoplakia with measurable clinical responses and favorable biomarker changes. Based on these results, we undertook a randomized, placebo controlled phase IIb trial with patients receiving BBIC or placebo for 6 months, with assessment of clinical response and change in lesion area as primary endpoint and an intent to treat analysis. 132 subjects were randomized; and 89 subjects completed six months on study drug or placebo. Both placebo and BBIC demonstrated a statistically significant decrease in mean lesion area of 17.1% and 20.6% respectively, and partial or greater clinical responses of 30% and 28% respectively. No significant difference between placebo and study drug arms was observed. Histological review, review of photographs of lesions, and comparison of serum neu protein and oral mucosal cell protease activity also did not show significant differences between study arms. Probable reasons for these negative results were considered and are discussed, and include a placebo with non-BBIC clinical activity and reduced pharmacokinetic availability of the second batch of BBIC. This experience should be a strong cautionary note to those considering “Green” Chemoprevention.

Introduction

Considerable epidemiological evidence links dietary habits and incidence of a variety of cancers. Consumption of high levels of soybeans has been associated with decreased incidence of cancer of the breast, colon, and prostate.[1-4] Soybeans contain a number of compounds that have potential anticarcinogenic activity including isoflavones, phytic acid, saponins, and several protease inhibitors. Both epidemiologic and experimental data strongly suggest a broad role for protease inhibitors in providing a protective effect against cancer formation, and those with chymotrypsin inhibitory (C.I.) activity have been found to be the most potent.[1, 2, 4, 5]

The Bowman Birk Inhibitor (BBI) is a serine protease inhibitor isolated from soybeans possessing domains with trypsin and chymotrypsin inhibitory activity.[6] In vitro and in vivo studies demonstrate anticarcinogenic activity in a number of animal model systems. BBI Concentrate (BBIC) has the same anticarcinogenic profile as purified BBI, and it has been developed for human trials.[5] Both BBI and BBIC are nontoxic, and safety has been reported in a phase I trial of BBIC administered as an oral troche in patients with oral leukoplakia.[4] BBIC safety has also been demonstrated in numerous other human trials utilizing BBIC.[7]

It has been suggested previously that premalignant human tissues may have elevated levels of proteolytic activity (PA) for hydrolysis of the tripeptide fluorescence substrate, butoxycarbonyl-valine-proline-arginine-(7-amino-4-methylcoumarin), which can be utilized as a biomarker for human cancer prevention studies).[8] This PA can be inhibited by several anticarcinogenic serine protease inhibitors, including BBI.[9] Since the abilities of these protease inhibitors to inhibit PA correlates with their abilities to suppress malignant transformation), [9] the hydrolysis activity may be involved in the carcinogenic process, and therefore, could be a candidate biomarker for human cancer prevention studies. Another biomarker potentially useful for human cancer prevention studies is neu oncoprotein.[10-13] Increased level of neu protein from tumor cells was shown to be correlated with the elevated concentration of neu protein in serum.[14, 15] Increased levels of neu protein have been detected as well in patients with various cancers, including cancers of the breast, ovary, prostate, stomach, pancreas, colon, liver, and lung.[10, 16, 17] In our phase IIa trial of BBIC modulation, neu protein levels were correlated with changes in protease activity in subjects receiving BBIC.[13, 18]

We have previously reported encouraging findings in a Phase IIa trial of BBIC in patients with oral leukoplakia.[4] Thirty one percent of 32 subjects receiving BBIC for one month demonstrated clinical response. A dose response relationship in decreased lesion size was observed, and blinded evaluation of lesion photographs confirmed this relationship. Protease activity was found to decrease in individuals with elevated activity at initiation of treatment. These encouraging results led us to proceed with a double-blind randomized-placebo controlled trial of BBIC in patients with oral leukoplakia for six months. Clinical and histological response, toxicity, and oral mucosal cell PA and Neu levels and serum Neu were evaluated.

MATERIALS AND METHODS

Design and Patient Characteristics

This was a two-armed, double-blind, placebo-controlled, multi-center, Phase II-B trial. There were eight geographically separated performance centers (in California, Massachusetts, New York, and Florida). Subjects had an equal chance of being assigned to the drug or placebo arm. Independent randomization schedules were created for each performance center (so study-arm assignment would not be confounded with geography). For those centers expected to accrue at a faster rate, the randomization schedule incorporated a block size of four. A block size of two was used for centers with lighter accrual goals.

The primary endpoint was relative percent change in total lesion area after six-months on study, and the percent of subjects showing a clinical response on that measure. Other endpoints included change in clinical impression from photographs of lesions, a central pathology review, and changes in buccal-cell and serum Neu protein, and buccal cell protease activity. Early in the study, subjects on the drug arm who showed a partial or complete response at six months were allowed to continue treatment for an additional 12 months (total 18 months) with final follow up at 21 months. However, due to the limited supply of drug the protocol was soon modified to limit treatment to six months.

Potential subjects were recruited through radio and print advertising, and outreach to dental associations. Eligible participants had to be at least 18 years old and have histologically proven oral leukoplakia and/or erythroplakia capable of being measured bi-dimensionally (to enable estimates of lesion area). Initially we required that total lesion area estimated at baseline be at least 100mm², but later this requirement was relaxed to facilitate accrual. Those otherwise meeting the inclusion criteria were excluded for any of the following reasons: 1) use of systemic or topical oral steroids within three months, 2) currently pregnant or lactating, 3) presence of head-and-neck cancer (including in-situ disease), or history of such within two years, 4) retinoid or beta-carotene therapy for any reason within two years, or beta-carotene capsules of any size within six months (patients were allowed up to 2 multi-vitamins per day), 5) participation in another randomized clinical trial within six months. Subjects had to be willing to have photographs taken of the lesion fields, and commit to serial appointments. Premenopausal and perimenopausal women were required to agree to employ adequate birth-control methods and have a negative pregnancy test. At the completion of the four-week run in phase of the study, 75% compliance with administration of drug measured by counting unused drug packets was required for continuation in the study. Written informed consent was obtained for all subjects at initial screening, and the study was approved by the institutional review boards of the University of California, Irvine and all other performance sites.

Study procedures

After consent and prescreening, potentially eligible subjects underwent a screening oral examination. Buccal mucosal cells were collected, urine specimen obtained, and approximately 15 ml. fasting blood drawn at the initial visit. If recent (within 3 months) histological analysis had not been documented with review of biopsy, 3 mm. punch biopsy of representative lesions were performed after measurement and photo-documentation of lesion(s).

Subjects meeting eligibility criteria underwent a one-month run-in period during which they self-administered the placebo compound. After the run-in period, if compliance with medication was adequate (see above), then baseline examinations were performed and biospecimens collected. Similar examinations were performed at three months and six months on treatment. Lesion biopsy and collection of blood and urine samples were repeated at the six-month visit. At three months off treatment, subjects were evaluated for adverse events and lesion measurements. Subjects were questioned about palatability, side effects, and any adverse events at each visit while on the trial.

Study Agent and Treatment

For the run-in interval, all patients received placebo, which was Quaker Tortilla Mix – Masa Harina De Maiz. During the run-in period only, 40 mg of citric acid per three grams Masa Harina was added for taste. This product was tested for chymotrypsin inhibitory activity and none was measurable. The dose level for placebo was six grams per day. Study drug was Bowman-Birk Inhibitor Concentrate (initially produced and supplied by Central Soya Company (Ft. Wayne, Indiana), but later supplied by the NIH/NCI/Division of Cancer Prevention pharmacy). Patients received daily doses of 600 C.I. units of BBIC, administered as 300 C.I. Units twice a day as 3 grams of BBIC or 3 grams of placebo. Both placebo and drug were provided to subjects as powder in small, zippered, amber, plastic pouches each containing one day’s dose. Participants were instructed to use half the material in the morning and the balance in the evening. Patients self-administered, by mixing the appropriate amount of powder with about 20 ml of water, swishing the mixture in the oral cavity for 60 seconds, then swallowing it. Treatment pouches were prepared centrally and sent to performance sites in lots of 100 pouches (3-month supply) per participant, renewed upon need.

Assessment of Clinical Response

The primary clinical response measure for each subject at each time point was estimated relative percent change in total lesion area, with primary interest being the comparison of baseline measures to those at six months on treatment. Clinical examiners recorded the estimated extent of the longest axis of each lesion and the extent perpendicular to the longest axis, in mm, at each time point for each subject. Clinicians made reference to previous exams to ensure all lesions were measured, recording values of zero for lesions that had resolved, and to document new lesions. After data entry, individual lesion areas were estimated by the product of length and width, and summed across lesions within each subject, by time point. Relative percent change in total lesion area was defined as 100 times (area post-treatment minus area pre-treatment) all divided by pre-treatment area. Category of clinical response was based on the magnitude of relative percent change in total lesion area. A complete response (CR) was declared if the relative percent change in total lesion area was minus 100 percent. A partial response (PR) was a relative percent decrease in total lesion area of 50 percent or more, without being a CR. Disease progression was a relative percent increase in total lesion area of at least 50 percent. Remaining cases were declared to be stable disease.

A secondary clinical response measure was based on blinded, comparative judgments of pairs of photographs of the same lesion at baseline and six-months on study. Study staff assembled all available pictures of lesions from pre-treatment (screening or randomization) and the six-month time points, by subject and lesion. Pictures at randomization were preferred over those at screening, but the latter were used absent the former. Any picture from one time point without a mate at the other time point was set aside. (Note: Lesions first appearing during the course of treatment had no mate from pre-treatment exams.) If a subject had multiple picture pairs for the same lesion, then one pair was chosen at random and the others set aside. In case of multiple pictures for a lesion at one time point, then one was picked at random for use and the others set aside. If there were picture pairs for more than one lesion for a particular subject, then one pair was selected at random for use and the others set aside. Thus, each subject having photos of the same lesion (or appropriate anatomic area, if the lesion had resolved) contributed exactly one picture pair to the task materials.

Picture pairs were arranged into a photo album, with one pair per page, one photo above the other. Picture pairs were assigned to album page at random, such that, both study performance center and treatment arm were balanced across the presentation sequence, as was whether the pre-treatment picture or the six-month picture was in the superior position. Five physicians experienced with evaluation of oral mucosal tissue abnormalities, but blinded to study arm and time point, independently compared the pictures in each pair using a seven-point scale. The scale ranged from, “Top photo shows a complete response relative to the bottom photo,” through, “The same degree of disease is shown by top photo and bottom photo,” to, “Bottom photo shows a complete response relative to the top photo.” Raw scores were transformed to account for relative position of the earlier and later photo, and averaged across the five reviewers. Final scores ranged from one, denoting a complete response at six-months, to four, which indicated no change, through seven, which indicated that the six-month photo depicted a much worse situation than the pre-treatment photo.

Pathologic Assessment

A single, experienced pathologist reviewed pre- and post-treatment pairs of tissue specimens. The reviewer was blinded to study-arm assignment (drug or placebo), but not to time point of specimen. For each specimen, the reviewer marked a continuum to indicate degree of tissue abnormality. The continuum was 140 mm long, and anchored by the word ‘Normal’ on the left and ‘Malignant’ on the right. The distance from the left edge of the continuum to the reviewer’s mark, in mm, was determined. For analyses, a score was formed by subtracting the pre-treatment value from the six-month value. Thus a retreat from ‘Malignancy’ over time produces a negative score, a score of zero denotes no change, and a positive score denotes a worsening situation.

The central pathologist also made a direct comparison of pre- and post-treatment specimens, marking a 170mm continuum anchored on the left by ‘Post treatment demonstrates no dysplasia in comparison to pretreatment,’ and on the right by, ‘Post-treatment demonstrates greater dysplasia than pretreatment.’ The center of the continuum was labeled, ‘Pretreatment and post treatment demonstrate no difference.’ The reviewer’s mark was coded as the distance from the left edge, in mm. On this measure low scores denote improvement over time, a score of 85 denotes no change, and a value higher than 85 indicates histological worsening.

Intermediate Marker Endpoints (IME)

Titer information was developed for Buccal-Cell Neu protein (ng/mg), Serum Neu protein (ng/ml), and Protease Activity (delta RFU / min / μg). Laboratory and assay methods were the same as described by Wan et al., 1999. Titer amounts by subject number and date were forwarded to the statistician, who consolidated the information. For all three IME, multiple readings for the same subject at the same time point were averaged. IME data are reported as the relative percent change from pre-treatment to six months (post minus pre, all divided by pre).

Statistical analyses and data management

Power estimates for our Phase IIB study were performed by the dose-response observed in our preceding, single-arm, Phase IIA trial in which study response rate (PR+CR) at the lowest dose (200 CIU) was 12.5 percent (1 out of 8 participants). We used this figure to anticipate response in the placebo arm of the Phase IIB effort. Similarly, we used the Phase IIA figure of 36.4 percent (4/11) responding (PR+CR) at the 533 CIU dose to anticipate the response rate of the treatment arm in the Phase IIB effort. Thus, the Phase IIB trial was powered to contrast response rates of about 12.5 and 36 percent, in both the Phase IIA and IIB trials; our criterion for a partial response was a reduction of estimated lesion area of at least 50 percent.

Analyses were performed using SAS/STAT® software.[19] Raw data were entered into a central database (Microsoft Access®) by staff of the Biostatistics Shared Resource of the UC Irvine Chao Family Comprehensive Cancer Center. Data on lesion size, and judgments of photos and pathology specimens were formed into SAS data sets, which were subject to 100 percent verification against the case-report forms by the study statistician. Results of IME assays were entered into spreadsheets by wet-lab staff, which the statistician converted to SAS data sets, which were then verified against the original spreadsheets. Data on host factors were extracted from the central database. Statistical tests of proportions were by Fisher’s Exact Test.[20]

Because measures of relative-percent change profoundly violate normality, as did estimates of total lesion area and the pathology judgments, comparisons of continuous measures between groups were by Wilcoxon two-sample test. [20] The signed-rank test was used to evaluate the null hypothesis that measures of change were centered on zero. [20] Confidence intervals about medians are based on order statistics. Correlations among relative percent change scores were by Spearman rank correlation. [21] No adjustment was made for multiple testing or for any correlation among the various dependent measures.

RESULTS

Participants

A total of 513 people were screened for participation across all eight performance sites, of which 188 (37%) were ineligible and 325 were consented. The data of first randomization was May 1999 and the last in September 2009. Of those screened, 118 (23%) did not complete the run-in period. Of those consented, 157 (48%) completed the run-in period but 25 declined to continue, resulting in 132 randomized patients. Sixty seven patients were randomized to the drug arm while 65 were assigned to placebo. The difference in numbers assigned to the two arms did not exceed one at any performance site. Of the 132 randomized, 89 participants (67.4%) had lesion sizes recorded at both randomization and six months, and so were evaluable on the primary endpoints. Twenty-one participants had lesion sizes recorded at randomization, but not at six months. A further 22 had no lesion measurements recorded at randomization (the majority from a remote performance site, with high turnover in study personnel).

The 89 subjects who were evaluable on the primary endpoint at six months seem fairly representative of the 132 who were randomized (Table 1). Importantly, the proportion of those randomized who contributed data at six months did not differ between study arms (drug arm 64% (43/67), placebo arm 71% (46/65), p > 0.46). Nor did that proportion differ by gender, (p > 0.44) or major race/ethnic group (p > 0.57). About 36% (48/132) of those randomized reported use of beer, wine, or liquor, while 42 percent (56/132) did not answer questions on alcohol consumption. Proportions reporting, not reporting, or denying alcohol use did not differ between those who did and did not contribute data on the primary endpoint at six months (p > 0.39). About 21 percent of those randomized (28/132) reported using tobacco (viz., cigarettes, cigars, pipe, oral use), while 50 percent (66/132) did not answer questions on tobacco use. Proportions reporting, not reporting, or denying tobacco use did not differ between those who did and did not contribute data on the primary endpoint at six months (p > 0.25). Estimates of total lesion area at randomization did not differ between those with lesion measurements only at randomization (n=21, mean 530.7mm², median 380mm², range 19-1976mm²) and those evaluable on the primary endpoint at six months (n=89, mean 502.6mm², median 310mm², range 48-3500mm²; p > 0.70).

Table 1.

Patient Characteristics By Study Arm For Those Evaluable on Relative Percent Change in Total Lesion Area at Six Months

Characteristic
	Overall	Drug	Placebo	Difference between arms
Number of Participants	89	43	46	%drop outs NS
Age
mean	60.7	58.7	62.6	NS
median	61.8	59.7	63.0
range	29-82	29:82	35:82
Sex (%male)	64%(57/89)	65%(28/43)	63%(29/46)	NS
Race/Ethnicity (%White)	78%(69/89)	33/43	36/46	NS
Alcohol Use (self report)
yes	35/89	16/43	19/46	NS
no	20/89	11/43	9/46
unknown	34/89	16/43	18/46
Tobacco Use (self report)
yes	20/89	12/43	8/46	NS
no	29/89	11/43	18/46
unknown	40/89	20/43	20/46
Total Lesion Area at Randomization
mean	502.6	492.4	512.1	NS
median	310	300	317.5
range	48-3500	48-3500	55-2550

Table 1 also summarizes measures describing the patients evaluable on the primary endpoint at six months, overall and by study arm. Fifty seven men and 32 women contributed data on relative percent change in total lesion area (the primary endpoint) at six months. The proportion that were male did not differ across study arm (p > 0.99), nor did the distribution of age (p > 0.15). About 78 percent (69/89) were White, and the proportion of Whites did not differ across study arm (p > 0.99). The proportions reporting, not reporting, or denying alcohol or tobacco use, did not differ between study arms (alcohol p > 0.82; tobacco: p > 0.29). Estimated total lesion area at randomization did not differ between study arms (p > 0.64).

Clinical Response

Table 2 shows the number and proportion showing various degrees of clinical response, by study arm. (Degree of clinical response is defined in the methods section, above.) Both the number and proportion of participants in each category of clinical response are similar across study arms (p > 0.94). About 28 percent of those in the drug arm achieved a partial response or better (12/43), while that figure for the placebo arm was 30 percent (14/46), which are not statistically different (p > 0.81). The two arms produced similar rates of disease progression: 9 percent in the drug arm (4/43) and 13 percent in the placebo arm (6/46) (p > 0.74). Thus the data in Table 2 show no evidence that the treatment produced a shift in the distribution of clinical response, relative to the placebo arm.

Table 2.

Number and Percent of Subjects by Category of Clinical Response at Six Months, by Study Arm

CLINICAL REPONSE	Drug	Placebo
Complete Response	2 (4.6%)	2 (4.4%)
Partial Response	10 (23.3%)	12 (26.1%)
Stable Disease	27 (62.8%)	26 (56.5%)
Disease Progression	4 (9.3%)	6 (13%)
Total	43 (100%)	46 (100%)

Complete Response: 100% reduction in total lesion areas. Partial Response: at least a 50% reduction, but not complete resolution, of total lesion areas. Stable Disease: Disease Progression: FET p > 0.90

Figure 1 shows the distribution of relative percent change in total lesion area after six months on treatment, by study arm, and Table 3 gives corresponding summary measures of these distributions. As the figure shows, the bulk of scores, ignoring study arm, fall below zero (sign-rank p < 0.0002) implying that lesion area was reduced in both groups. Per Table 3, the median relative percent change across study arms was about a 26-percent reduction. The distributions by study arm appear reasonably coincident, with no sign of a difference in location (p > 0.75) or spread (F(45,42)=1.60, p > .12).

Figure 1. Relative Percent Change in Total Lesion Area, by Study Arm.

Legend:BBIC: N=43, Median=−16 (grey bars)

Placebo: N=46, Median=−26.5, sd=66 (black, solid bars)

Total: N=89, Median=−26.5, sd=60,

Range: −100 to 219.5

Median test, p > 0.45

TABLE 3.

Summary of Relative Percent Change in Total Lesion Area from Baseline to Six-Months on Study, by Study Arm

STUDY ARM	N	MEAN	SD	MEDIAN	Smallest	Largest
Drug	43	−20.6	52.4	−16.5	−100.0	+123.5
Placebo	46	−17.1	66.4	−26.5	−100.0	+219.5
TOTAL	89	−18.8	59.7	−26.5	−100.0	+219.5

Wilcoxon two−sample test, p > 0.75

Clinical Impression from Photographs Confirmed these Assessments (Supplemental A). No significant adverse events related to drug or placebo administration were observed during the trial (details Supplemental B). This assessment included the 10 patients treated with BBIC or placebo for up to 18 months in the trial. Medication was tolerated, with some subjects expressing palatability issues. The distribution of adverse events in the 2 arms was not different. Pre and post levels of cholesterol and tryglycerides was similar in the two groups (Supplemental C).

Central Pathologic Review

The central pathologist reviewed 98 pairs of slides, a small percentage of which were re-cuts substituted for original material that could not be evaluated. Three subjects had materials for multiple lesions, but for analysis we randomly selected just one lesion in each case. Each participant contributed just one lesion to the analysis. One subject was dropped because the only available post-randomization specimen was from the three-month time point. The materials for an additional four subjects were deemed inadequate for review, and no re-cuts were available. Thus final analysis was based on 88 subjects, representing 67 percent of those randomized (88/132). This proportion did not differ statistically across study arm (drug: 41/67; placebo: 47/65; p > 0.19).

Table 4 summarizes the difference in rated degree of malignancy between randomization and six-month specimen. Positive values indicate histologic worsening while negative scores denote improvement over the six-month study period. Across study arms, the location of these scores is not statistically different from zero (sign-rank, p > 0.46), and, consistent with inspection of the table, there is no difference between groups (p > 0.88). A similar result was obtained from the pathologist’s direct comparison of the specimens. The median score overall and for each study arm was that point indicating no change, and of course there is no significant difference between the two arms (p > 0.68: data not shown).

Table 4.

Summary Measures of the Difference in Rating of Degree of Malignancy of Tissue from Pre-Treatment to Six-Months on Study, by Study Arm

Study Arm	N	Mean	SD	Median	Smallest	Largest
Drug	41	1.2	23.7	0	−86	71
Placebo	47	3.6	15.4	0	−20	59
TOTAL	88	2.5	19.6	0	−86	71

Negative scores indicate a change toward normal tissue. Positive scores indicate a change toward malignancy. Values of zero indicate no change.

Intermediate Markers

Table 5 shows relative percent change over six months on-study in buccal-cell Neu protein and protease activity, and serum-Neu protein, overall and for each study arm. Necessary specimens and evaluable results for these assays were available for 45, 45, and 41 subjects, respectively. Pooled across study arm, the relative percent change in protease activity was statistically elevated above zero (sign-rank, p < 0.05, n=45). However, neither study arm demonstrated this effect when considered separately (placebo: p > 0.07; drug: p > 0.30). Relative percent change in neither of the remaining two markers was statistically different from zero, either overall (ps > 0.18) or by study arm (ps > 0.30). None of the three measures showed a statistically significant difference between placebo and drug arms (ps > 0.32). When study arm was ignored, none of these three measures showed a significant correlation with relative percent change in total lesion area (buccal-cell Neu: rank correlation 0.11, p > 0.45, N=45; protease activity: rank correlation 0.92, p > 0.88, N=45; serum Neu: rank correlation 0.07, p > 0.66, N=41).

Table 5.

Median Relative Percent Change in Biomarkers, by Study Arm

ARM:	Overall		Placebo		Drug
MARKER	median	95% CI	median	95% CI	median	95% CI
Buccal-Cell Neu	−8.9 N=45	−24.5 +18.5	−4.2 N=24	−33.6 +36.7	−10.1 N=21	−52.3 +24.6
Protease Activity	+16.2 N=45	−10.0 +27.0	+17.2 N=24	−10.3 +30.9	+15.7 N=21	−23.8 +74.9
Serum Neu	−4.1 N=41	−11.3 +0.7	−8.1 N=22	−15.3 +6.6	−3.9 N=19	−13.5 +8.1

DISCUSSION

In this phase IIb clinical trial of BBIC, no significant difference between placebo and treatment arms was identified for clinical response, change in lesion area, histologic grade, or biomarkers studied. Interestingly, both the treatment and placebo group showed statistically significant decreases in total lesion area (Table 3), and the two groups demonstrated partial + complete response rates of 27.9% and 30.5% respectively (Table 2). No significant difference in photographic review of lesions was identified between study groups, but the placebo group showed slight improvement following treatment. Oral leukoplakia has a waxing and waning clinical course, and spontaneous improvement or disappearance has been reported in over 10% of cases.[22, 23] However, the approximate 30% response rate raises the possibility that there was clinical activity in the placebo used in the trial.

In designing the study, considerable attention was directed at placebo selection. It was very difficult to identify a compound that would be inert, palatable and have similar consistency and physical properties similar to those of BBIC. Ultimately, a corn based product was selected, and Masa Harina was used. Although no chymotrypsin inhibitory activity was measurable, this product contains several vitamins, antioxidants, protease inhibitors and supplemental nutrients, and is processed by lime cooking, which alters levels of phytochemicals and phenolic compounds, such as ferulic acid. [24] The levels of calcium and ferulic acid were sufficiently high in the placebo that cancer preventive activity may have been present; in addition, several other nutrients were present in the placebo at micromolar concentrations that have been shown to have radioprotective or cancer preventive activity in other studies (as described in Supplemental D). These properties raise the issue of the placebo having had an unexpected clinical effect. Similar concerns about a placebo effect have been raised in a trial of the cyclooxygenase inhibitor Ketorolac as an oral rinse in oropharyngeal leukoplakia (25), suggesting that the placebo oral rinse, which contains alcohol, and improved oral hygiene may contribute to a beneficial overall effect on oral inflammatory processes. In studies of other oral inflammatory processes, higher than expected response rates were attributed to increased frequency of oral rinsing (26, 27). In total, this report indicates that future trials of chemoprevention agents in oral leukoplakia need to carefully consider whether a no treatment arm should be part of the trial and/or a much larger trial should be done. However, with a 30 percent placebo effect, trials become prohibitively large (see Supplemental F for discussion).

The potential activity of the placebo raises concerns about the overall result. A number of natural compounds have potential chemopreventive activity, and if these compounds are biologically active, they will mask effects of the study drug tested in placebo controlled trials. Even limited activity could mask the clinical effect of the study drug, and require larger sample sizes to distinguish statistically significant differences in clinical effect. Given the difficulty of accruing subjects in chemoprevention studies, this will make these studies even more expensive and prolonged. This dilemma has raised the question of whether alternative study designs would be more effective. For example, a study design in which the patient serves as her/his own control with serial periods on or off study drug may eliminate issues related to placebo activity.

To further complicate the evaluation, there appears to have been a loss in drug potency over time. An interim analysis was performed approximately half way through the study. Preliminary blinded analysis indicated a modest but promising difference in treatment response between the two study arms (Supplemental E). Based on these encouraging results, the trial was continued and completed. Analysis of the subjects accrued during the second half of the trial demonstrated no difference between the two arms of the study. These results suggest that patients treated with BBIC in the earlier years of the trial exhibited better responses, in terms of effects on size of the oral leukoplakia lesions as well as the intermediate marker endpoints evaluated, than those treated with BBIC in the later trial years, (as detailed in Supplemental E). It is hypothesized that the apparent loss of potency could have been caused by the BBI storage conditions during this long time period. The directions that came with packages of BBIC initially prepared by Central Soya Company (and dispensed during the early years of the oral leukoplakia trial) indicated that BBIC should not be refrigerated or frozen, as it was known at an early stage of BBIC development that storage under refrigerated or frozen conditions greatly diminished the ability of BBIC to inhibit the malignant transformation of cells in vitro. [28] Retrospectively, it was discovered that a different batch of BBIC (termed the new formulation of BBIC), maintained by the same pharmacy that dispensed BBIC during the later years of the oral leukoplakia trial, was kept at both refrigerated and frozen temperatures. Analysis of the bioavailability of the original and new formulations of BBIC in human subjects demonstrated an approximately 60% reduction in bioavailability of the new formulation, compared to the original formulation, of BBIC (unpublished results, made available to the reviewers). As the new formulation of BBIC was maintained under both refrigerated and/or frozen conditions by the same pharmacy that provided the BBIC for the oral leukoplakia trial, it is conceivable that storage of BBIC in a refrigerated/frozen state could have decreased the clinical activity of the drug during the later years of the oral leukoplakia trial. The reduced bioavailability of the second BBIC product also raises concerns as our Phase IIA trial demonstrated a clear dose-response effect (4) and a 40 percent bioavailability would be below the effectiveness of the chosen dose in the current Phase IIB trial. Another difficulty encountered was the long time duration between trial initiation and completion; oral leukoplakia patients were actually treated with BBIC over a period of ~11 years (May 1999 - March, 2010).

In conclusion, this phase IIb randomized placebo controlled trial did not show greater efficacy for the BBIC compared to placebo in patients with oral leukoplakia. This experience should be a strong cautionary note to those considering “Green” Chemoprevention. [29]