Topical application of a mucoadhesive freeze-dried black raspberry gel induces clinical and histologic regression and reduces loss of heterozygosity events in premalignant oral intraepithelial lesions: results from a multicentered, placebo-controlled clinical trial.

PURPOSE: Approximately 30% higher grade premalignant oral intraepithelial neoplasia (OIN) lesions will progress to oral cancer. Although surgery is the OIN treatment mainstay, many OIN lesions recur, which is highly problematic for both surgeons and patients. This clinical trial assessed the chemopreventive efficacy of a natural product-based bioadhesive gel on OIN lesions. EXPERIMENTAL DESIGN: This placebo-controlled multicenter study investigated the effects of topical application of bioadhesive gels that contained either 10% w/w freeze-dried black raspberries (BRB) or an identical formulation devoid of BRB placebo to biopsy-confirmed OIN lesions (0.5 g × q.i.d., 12 weeks). Baseline evaluative parameters (size, histologic grade, LOH events) were comparable in the randomly assigned BRB (n = 22) and placebo (n = 18) gel cohorts. Evaluative parameters were: histologic grade, clinical size, and LOH. RESULTS: Topical application of the BRB gel to OIN lesions resulted in statistically significant reductions in lesional sizes, histologic grades, and LOH events. In contrast, placebo gel lesions demonstrated a significant increase in lesional size and no significant effects on histologic grade or LOH events. Collectively, these data strongly support BRB's chemopreventive impact. A cohort of very BRB-responsive patients, as demonstrated by high therapeutic efficacy, was identified. Corresponding protein profiling studies, which demonstrated higher pretreatment levels of BRB metabolic and keratinocyte differentiation enzymes in BRB-responsive lesions, reinforce the importance of local metabolism and differentiation competency. CONCLUSIONS: Results from this trial substantiate the LOH reductions identified in the pilot BRB gel study and extend therapeutic effects to significant improvements in histologic grade and lesional size.

Distribution of anthocyanins delivered from a bioadhesive black raspberry gel following topical intraoral application in normal healthy volunteers.

PURPOSE: Results from our oral cavity chemoprevention trial demonstrated appreciable interpatient variations regarding chemopreventive efficacy of a freeze dried black raspberry (FBR) gel. We speculated these data reflected individual patient-related differences in absorption, target tissue uptake and local compound metabolism of key FBR compounds (anthocyanins). Accordingly, this study assessed the distribution of anthocyanins from the 10% (w/w) FBR gel in saliva, oral tissues and plasma. METHODS: Human subject participation entailed collection of: (1) saliva, tissue and plasma (5 min following gel application, keratinized tissues), (2) saliva and plasma (5 min after sublingual gel application), (3) saliva and plasma at 1, 2, and 4 h post gel application (keratinized tissues), and (4) saliva (cyanidin 3-rutinoside incubations). Levels of FBR anthocyanins in the respective samples were analyzed by LC/MS/MS. RESULTS: Our data show: significantly higher anthocyanin levels in saliva and oral tissues relative to matched plasma samples, marked donor-specific variations in anthocyanin uptake, sustainability of anthocyanins at the target site, pH affects anthocyanin penetration and intraoral anthocyanin decomposition and/or metabolism. CONCLUSIONS: No previous oral cavity chemoprevention trials evaluated compound distribution at the treatment site. Our data, which demonstrate a local delivery-derived pharmacologic advantage, provide insights which could advance oral cavity chemoprevention strategies.

Topical application of a bioadhesive black raspberry gel modulates gene expression and reduces cyclooxygenase 2 protein in human premalignant oral lesions.

Reduced expression of proapoptotic and terminal differentiation genes in conjunction with increased levels of the proinflammatory and angiogenesis-inducing enzymes, cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS), correlate with malignant transformation of oral intraepithelial neoplasia (IEN). Accordingly, this study investigated the effects of a 10% (w/w) freeze-dried black raspberry gel on oral IEN histopathology, gene expression profiles, intraepithelial COX-2 and iNOS proteins, and microvascular densities. Our laboratories have shown that freeze-dried black raspberries possess antioxidant properties and also induce keratinocyte apoptosis and terminal differentiation. Oral IEN tissues were hemisected to provide samples for pretreatment diagnoses and establish baseline biochemical and molecular variables. Treatment of the remaining lesional tissue (0.5 g gel applied four times daily for 6 weeks) began 1 week after the initial biopsy. RNA was isolated from snap-frozen IEN lesions for microarray analyses, followed by quantitative reverse transcription-PCR validation. Additional epithelial gene-specific quantitative reverse transcription-PCR analyses facilitated the assessment of target tissue treatment effects. Surface epithelial COX-2 and iNOS protein levels and microvascular densities were determined by image analysis quantified immunohistochemistry. Topical berry gel application uniformly suppressed genes associated with RNA processing, growth factor recycling, and inhibition of apoptosis. Although the majority of participants showed posttreatment decreases in epithelial iNOS and COX-2 proteins, only COX-2 reductions were statistically significant. These data show that berry gel application modulated oral IEN gene expression profiles, ultimately reducing epithelial COX-2 protein. In a patient subset, berry gel application also reduced vascular densities in the superficial connective tissues and induced genes associated with keratinocyte terminal differentiation.

Effects of a topically applied bioadhesive berry gel on loss of heterozygosity indices in premalignant oral lesions.

PURPOSE: The aim of this study was to assess the effects of topical application of a 10% (w/w) freeze-dried black raspberry (FBR) gel on oral intraepithelial neoplasia (IEN) variables that included histologic diagnoses and loss of heterozygosity (LOH) indices. Microsatellite instability and/or LOH at tumor suppressor gene-associated chromosomal loci have been associated with a higher risk for oral IEN progression to oral squamous cell carcinoma. Previously, our laboratories have shown that FBRs are well tolerated and possess potent antioxidant, apoptotic, and differentiation-inducing properties. EXPERIMENTAL DESIGN: Each participant with IEN served as their own internal control. Before treatment, all lesions were photographed, and lesional tissue was hemisected to obtain a pretreatment diagnosis and baseline biochemical and molecular variables. Gel dosing (0.5 g applied four times daily for 6 weeks) was initiated 1 week after the initial biopsy. Genomic DNA was isolated from laser-captured basilar and suprabasilar surface epithelial cells followed by PCR amplification using primer sets that targeted known and presumed tumor suppressor gene loci associated with INK4a/ARF, p53, and FHIT. Allelic imbalance was determined by sequence analysis using normal participant tissues to establish microsatellite marker peak patterns and allele sizes. RESULTS: Confirming earlier phase I data, none of the 27 participants developed FBR gel-associated toxicities. Furthermore, our results show histologic regression in a subset of patients as well as statistically significant reduction in LOH at tumor suppressor gene-associated loci. CONCLUSIONS: These preliminary data suggest that further evaluation of berry gels for oral IEN chemoprevention is warranted.

Formulation and in-vitro and in-vivo evaluation of a mucoadhesive gel containing freeze dried black raspberries: implications for oral cancer chemoprevention.

PURPOSE: The purpose of these studies was to formulate mucoadhesive gels containing freeze dried black raspberries (FBR) and to determine optimum parameters for a subset of FBR bioactive compounds including anthocyanin stability, absorption and penetration in-vitro and in-vivo. MATERIALS AND METHODS: Berry gels were prepared having FBR at 5% and 10% w/w and final pHs ranging from 3.5 to 7.5. A HPLC assay was developed to quantify and determine the stability of the anthocyanins in the gels. A single time-point study was performed to determine anthocyanin uptake when the gels were applied to oral mucosa. Penetration of anthocyanins into human oral tissue explants was determined as a function of gel pH and FBR content. A HPLC-mass spectroscopy assay was utilized to quantify the anthocyanin levels in human oral tissue explants, saliva, and blood. RESULTS: The stability of anthocyanins in the gel was directly related to gel pH and storage temperature. Maximum stability of anthocyanins was found at lower pH (pH 3.5) and storage temperature (4 degrees C). Anthocyanins contained in mucoadhesive berry gel formulations were readily absorbed into human oral mucosa tissue as evidenced by detectable blood levels within 5 min after gel application. There was a trend for greater penetration of anthocyanins into tissue explants for berry gels with a final pH of 6.5 versus pH 3.5. CONCLUSIONS: Formulation and characterization of a novel gel formulation for local delivery of chemopreventive compounds to human oral mucosal tissues has been described. The results show anthocyanin stability was dependent upon gel pH and storage temperature and also demonstrate that the gel composition is well-suited for absorption and penetration into the target oral mucosal tissue site.

Suppression of the tumorigenic phenotype in human oral squamous cell carcinoma cells by an ethanol extract derived from freeze-dried black raspberries.

Despite focused efforts to improve therapy, 5-yr survival rates for persons with advanced-stage oral squamous cell carcinoma (SCC) remain discouragingly low. Clearly, early detection combined with strategies for local intervention, such as chemoprevention prior to SCC development, could dramatically improve clinical outcomes. Previously conducted oral cavity human chemoprevention trials, however, have provided mixed results. Although some therapies showed efficacy, they were often accompanied by either significant toxicities or circulating antiadenoviral antibodies. It is clearly apparent that identification of nontoxic, effective treatments is essential to prevent malignant transformation of oral epithelial dysplasias. This study employed cell lines isolated from human oral SCC tumors to investigate the effects of a freeze-dried black raspberry ethanol extract (RO-ET) on cellular growth characteristics often associated with a transformed phenotype such as sustained proliferation, induction of angiogenesis, and production of high levels of reactive species. Our results demonstrate that RO-ET suppresses cell proliferation without perturbing viability, inhibits translation of the complete angiogenic cytokine vascular endothelial growth factor, suppresses nitric oxide synthase activity, and induces both apoptosis and terminal differentiation. These data imply that RO-ET is a promising candidate for use as a chemopreventive agent in persons with oral epithelial dysplasia.