Brynmor A. Watkins

Bony changes in the jaws of rats treated with zoledronic acid and dexamethasone before dental extractions mimic bisphosphonate-related osteonecrosis in cancer patients

Osteonecrosis of the jaw is associated with aminobisphosphonate use in patients treated with intravenous doses for the prevention of bony metastases. A more complete understanding of the natural history of bisphosphonate-related osteonecrosis of the jaws (BRONJ), factors associated with risk, and its pathobiology has been limited by the availability of human material and the absence of clinical predictability. We now describe an animal model, developed in female Sprague-Dawley rats, in which we replicate many of the clinical, radiographic, and histologic features described in humans. Animals treated with a sequence of zoledronic acid (ZA) and dexamethosone (DX) over a one to three week period developed BRONJ-like changes following extraction of mandibular or maxillary molars. Whereas the extraction sites of control animals underwent predictable healing with rapid epithelialization, animals treated with ZA/DX demonstrated clinical and histological evidence of ulceration overlying areas of necrotic bone. In contrast to images from control animals, radiographs from animals treated with ZA/DX demonstrated poor definition of the alveolar ridge with mixed radiodensity. Modest increases in the extent of the inflammatory infiltrate were seen fourteen days after extraction in ZA-only treated animals compared to control or ZA/DX-treated rats. However, by post-extraction day 28, no differences were observed. Tissue vascularity was most pronounced in ZA-only treated animals compared to ZA/DX or control specimens. Apoptosis of epithelial cells was not observed in any experimental groups, and no evidence of Actinomyces was observed as determined by Periodic Acid Schiff (PAS) staining. The administration of ZA/DX preceding dental extractions in rats therefore results in the development of bony and soft tissue changes that are similar to those noted humans who develop BRONJ, and may provide a useful model for study of its pathogenesis, as well as strategies for its prevention and treatment.

AG013, a mouth rinse formulation of Lactococcus lactis secreting human Trefoil Factor 1, provides a safe and efficacious therapeutic tool for treating oral mucositis

Non-clinical studies, focusing on the pharmacodynamics (PD), pharmacokinetics (PK) and safety pharmacology of genetically modified Lactococcus lactis (L. lactis) bacteria, engineered to secrete human Trefoil Factor 1 (hTFF1), were performed to provide proof-of-concept for the treatment of oral mucositis (OM) patients. L. lactis strain sAGX0085 was constructed by stably inserting an htff1 expression cassette into the bacterial genome, and clinically formulated as a mouth rinse (coded AG013). PD studies, using different oral dosing regimens, were performed in a clinically relevant hamster model for radiation-induced OM. The PK profile was assessed in healthy hamsters and in hamsters with radiation-induced OM. In addition, in vitro and in vivo safety pharmacology studies were conducted, in pooled, complement-preserved human serum, and in neutropenic hamsters and rats respectively. Topical administration of L. lactis sAGX0085/AG013 to the oral mucosa significantly reduced the severity and course of radiation-induced OM. PK studies demonstrated that both living L. lactis bacteria, as well as the hTFF1 secreted, could be recovered from the administration site for maximum 24h post-dosing, without systemic exposure. The in vitro and in vivo safety pharmacology studies confirmed that L. lactis sAGX0085 could not survive in systemic circulation, not even under neutropenic conditions. The results from the PD, PK and safety pharmacology studies reported here indicate that in situ secretion of hTFF1 by topically administered L. lactis bacteria provides a safe and efficacious therapeutic tool for the prevention and treatment of OM.

Efficacy of Superoxide Dismutase Mimetic M40403 in Attenuating Radiation-Induced Oral Mucositis in Hamsters

Purpose: M40403 is a small-molecule superoxide dismutase mimetic that has shown efficacy in animal model disease states in which superoxide anions are thought to play a key role. Radiation treatment and chemotherapy for cancer generate free oxygen radicals that are hypothesized to trigger unwanted side effects in healthy tissue. For some patients undergoing these antineoplastic treatments, one of the most prevalent side effects is oral mucositis, which is a painful, often dose-limiting condition. Preclinical and clinical studies of this condition have shown the positive effect of treatment with compounds that decrease free oxygen radicals. This study investigated the efficacy M40403 in a clinically relevant hamster model of acute, radiation-induced oral mucositis. Methods: Oral mucositis was induced in hamsters by irradiation of the cheek pouch. The ability of i.p. administered M40403 to decrease the duration and severity of oral mucositis was assessed after treatment at different doses and dosing schedules. Oral mucositis was scored using the WHO grading scale. Results: Compared with placebo-treated animals, those irradiated on day 0 and treated twice daily with 30 mg/kg M40403 had significantly less severe and shorter duration mucositis over a range of treatment schedules, including from days -1 to 3, day 0 to 3, and day 0 alone. Similar efficacy was achieved at doses of 10 and 3 mg/kg twice daily on days -1 to 3. Conclusions: These results implicate free oxygen radicals in the onset of oral mucositis and also provide the basis for further development of M40403 in the prevention of this condition in at-risk cancer patients.

Serum amyloid P ameliorates radiation-induced oral mucositis and fibrosis

PurposeTo evaluate the effect of the anti-fibrotic protein serum amyloid P (SAP) on radiation-induced oral mucositis (OM) and fibrosis in a hamster cheek-pouch model.Experimental DesignHamsters received a single dose of radiation (40 Gy) to the left everted cheek pouch to induce significant OM. The protective therapeutic potential of SAP was evaluated using varying dosing regimens. The extent of OM was measured using a validated six-point scoring scheme ranging from 0 (normal tissue, no mucositis) to 5 (complete ulceration). Fibrotic remodeling was also visualized histologically and quantified at later time points using collagen gene expression.ResultsSAP treatment attenuated the profile of radiation-induced oral mucositis by delaying the time of onset, reducing the peak value, and enhancing the resolution of injury. The peak mucositis score was reduced by approximately 0.5 grade in SAP-treated animals. The number of animal days with a score of ≥ 3 was reduced by 48% in the SAP-treated group, compared with the saline control group (P < 0.01). SAP also inhibited the extent of tissue remodeling and decreased radiation-induced increases in myofibroblast number. Attenuated collagen deposition and gene expression was also observed in the cheek pouches of hamsters treated with SAP at both 16 and 28 days post-radiation.ConclusionsSAP treatment significantly attenuated radiation-induced injury. In particular, SAP attenuated the severity of OM and inhibited pathogenic remodeling. This suggests that SAP may be a useful therapy for the palliation of side effects observed during treatment for head and neck cancer.

Velafermin (rhFGF-20) reduces the severity and duration of hamster cheek pouch mucositis induced by fractionated radiation

Purpose: Velafermin (recombinant human fibroblast growth factor-20, rhFGF-20) has been shown to reduce the severity and duration of mucositis in preclinical acute (single dose) radiation and chemotherapy/radiation models of oral mucositis. Our present study assessed the impact of velafermin on the severity and duration of oral mucositis that occurred as a consequence of fractionated radiation. Experimental design: Male Golden Syrian hamsters were exposed to eight doses of radiation (7.5 Gy/dose) to the cheek pouch on days 0, 1, 2, 3, 6, 7, 8 and 9 that resulted in severe mucositis. Velafermin (4 mg/kg intraperitoneally) was administered on days 3 and 9; days 2, 3, 8 and 9; days 3, 4, 9 and 10; or days 4, 5, 10 and 11. Results: Although all velafermin-treated groups showed some reduction in the degree of mucositis relative to the vehicle control, the most significant reduction (p < 0.001) was observed in the groups treated on days 3 and 9 or on days 4, 5, 10 and 11. Further histological analysis of resected buccal mucosa revealed improvements in epithelial tissue degradation, connective tissue degradation and inflammation severity after velafermin treatment. Most notably, velafermin treatment reduced inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor (TNF) production possibly through nuclear factor-κB (NF-κB) mediation. The detection of increased NF-E2-related factor-2 (NRF-2) expression in the early onset stage of mucositis in the buccal mucosa suggested additional protective benefits from reactive oxygen species (ROS) generated as a consequence of fractionated radiation treatment. Conclusion: Thus, velafermin provided therapeutic benefit in a hamster model of oral mucositis induced by fractionated radiation therapy.

Attenuation of radiation‐ and chemoradiation‐induced mucositis using gamma‐d‐glutamyl‐l‐tryptophan (SCV‐07)

OBJECTIVE To evaluate the efficacy of a novel immunomodulating peptide (SCV-07) in attenuating the course of radiation-induced mucositis in an established animal model of oral mucositis (OM). MATERIAL AND METHODS In three separate experiments, golden Syrian hamsters received either an acute radiation challenge to the buccal mucosa of eight fractionated doses of 7.5 Gy of radiation over a 2-week-period, or a combination of acute radiation and cisplatin. In each experiment, animals were treated with varying doses or schedules of SCV-07 or placebo. OM was scored in a blinded fashion using digital images obtained during the experimental period. RESULTS We found that SCV-07 reduced the severity and duration of both acute and fractionated radiation-induced OM. Similarly, when radiation and chemotherapy were used to induce OM, treatment with SCV-07 significantly reduced the duration of ulcerative OM. The therapeutic benefit was dependent on both dose and schedule of administration. CONCLUSION Taken together, we found SCV-07 was able to modify the duration and severity of oral mucositis and was dependent on schedule and dose.

Single-Dose Prevention or Short-Term Treatment with Fibroblast Growth Factor-20 (CG53135-05) Reduces the Severity and Duration of Oral Mucositis

Oral mucositis (OM) is a treatment-limiting condition associated with myelosupressive chemotherapy and radiation therapy. The objective of this study was to evaluate the activity of recombinant human fibroblast growth factor-20 (FGF-20 or CG53135-05) in the prevention and treatment of OM in experimental animals. Oral mucositis was induced in hamsters with 5-fluorouracil (5-FU; 60 mg/kg) on days -4 and -2, followed by targeted irradiation with 30 Gy on day 0. Oral mucositis was scored every other day using severity scores of 0-5. To test for prevention of OM, animals received varying doses of FGF-20 on day 1 or days 1 and 2 after irradiation before the development of symptoms. To test the effects of FGF-20 on established mucositis, animals were allowed to develop early OM (score of 2) before treatment initiation. Animals then received FGF-20 by intraperitoneal (i.p.) administration (12 mg/kg) for 1, 2, 3, or 4 consecutive days. When prevention of OM was tested, administration of FGF-20 (12 mg/kg i.p.) on day 1 or days 1 and 2 resulted in significant reduction in duration of severe OM to 26% (P < 0.003) or 29.4% (P <0.018) of cumulative days, respectively, compared with untreated control animals, which spent 40.2% of cumulative days with OM scores >/= 3. When the effects of FGF-20 on established mucositis were tested, treatment of animals with FGF-20 for 2, 3, or 4 consecutive days resulted in significant reduction of severe OM to 27.4%, 29.2%, or 18.5% of days, respectively, compared with vehicle-treated control animals, which spent 41.1% of cumulative days with OM scores >/=3 (P < 0.05). These findings support the utility of FGF-20 as a single-dose agent in the prevention of OM. In addition, the positive effects of FGF-20 on established mucositis may permit treatment of patients with OM who may not benefit from prophylactic agents.

Abstract 2181: Rapid screening of molecular targets using RT-PCR for solid tumor profiling

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Purpose: The identification of molecular targets will be an essential step in developing mechanistically-based approaches to cancer treatment, as well as for determining tumor response and/or resistance to therapy. Not only will identification of these targets in solid tumors, but in the subsequent metastasis as well. The purpose of this study was to evaluate the utility of a large-scale tumor bank, using both xenograft and orthotopic models, as a source of material for quantification of gene expression as a tumor profiling and screening tool. Experimental Design: Solid tumors representing nine different indications were grown as a xenograft in nude mice. The panel of cell lines included: epidermoid carcinoma (A-431), lung carcinoma (A549, NCI-H460, small cell lung carcinoma: NCI-H82, NCI-H69), pancreatic carcinoma (AsPC-1, BxPC-3 and PANC-1), prostate carcinoma (Du145), breast carcinoma (MDA-MB-231, MDA-MB-435, MCF-7), colon carcincoma (HCT-15, HCT-116 and HT-29), ovarian carcinoma (OVCAR-3 and SK-OV-3) and glioma (U87-MG and U118-MG). In addition, five cell lines representing three indications were grown orthotopically (A549, NCI-H460, MDA-MB-231, MCF-7 and HT-29) in the lung, breast or colon, respectively. When subcutaneously implanted tumors reached a volume of 1500-2000mm3 samples were harvested by aseptic technique. Orthotopically implanted tumors were excised after 25 to 45 days post-implantation and tissues harvested. Animals with mammary fat pad tumors, also had lymph nodes and lungs removed. RNA was purified under sterile conditions. Expression of Akt, phosphoinositide-3 kinase (PI3K), epidermal growth factor receptor (EGFR), p53 and BCL-2 was determined using RT-PCR (Applied BioSystems TaqMan System). Results: Differential expression of target genes within solid tumors was determined using RT-PCR. An emphasis on gene expression in orthotopic and metastatic models will be discussed. Conclusion: Unlike semi-quantitiative methods such as immunohistochemistry (IHC) or Western Blotting, RT-PCR is ideal as screening tool as it is rapid, robust, quantitative and only requires a small amount of starting material. We describe a rapid screening method of gene expression in both xenograft and orthotopic models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2181.

Abstract A28: A murine model for cancer treatment‐related fatigue

Objectives: Among patients being treated for cancer, cancer treatment‐related fatigue (CTRF) remains a poorly understood condition with a profound effect on quality of life. Our objective was to develop a murine model of CTRF, independent of anemia, in order to further understand its pathogenesis and provide a conduit for the development of mechanistically‐based therapeutic approaches. Methods: To determine the optimal dose of ionizing radiation to produce fatigue, female BALB/c mice were irradiated with 10 doses of fractionated total body irradiation (fTBI, 8, 9, and 10 Gy, cumulative dose) or 15 doses (fTBI, 10.5 and 13.5 Gy, cumulative dose). In a separate experiment, mice received 70, 60 or 50 mg/kg of etoposide sulfate to determine the optimal dose of chemotherapy to induce CTRF. Cage‐top activity sensors (Phillips Respironics) were used to evaluate daily activity patterns in animals up to 4 weeks following the completion of radiation or 12 weeks following etoposide sulfate administration. CTRF was induced by 15 doses of fractionated total body irradiation (fTBI, 10.5 Gy, cumulative dose) in female BALB/c mice or 60mg/kg etoposide sulfate administered as a single dose. Total daily animal activity and changes in wake/sleep cycles were compared to baseline levels. Systemic levels of IL‐6 and TNF‐α were measured and anemia and leukopenia were assessed using specimens obtained in the weeks following the last dose of radiation or etoposide sulfate administration. Results: fTBI resulted in animals with a significantly lower cumulative daily activity, disrupted sleep/wake cycles that continued up to forty days following the last dose of radiation. Administration of etoposide sulfate resulted in a significantly lower cumulative daily activity three weeks following chemotherapy administration and continued for the duration of the study. Reduced activity levels were not associated with laboratory parameters suggestive of anemia. Changes in the measured hematological parameters demonstrated a temporal, dose‐dependent and reversible pattern; consistent with those previously reported in human patients. Conclusions: Despite its frequency and impact, CTRF has been accepted as a cost of successful cancer treatment. This conclusion was largely based on the supposition that, aside for symptom control, there was no opportunity to develop an effective intervention. We report the development a novel mouse model of CTRF in which administration of fractionated TBI or etoposide sulfate was able to induce behaviors consistent with fatigue reported in humans. Our data supports the hypothesis that TBI and chemotherapy induce systemic effects that result in changes in daily activity patterns and sleep/wake cycles in the absence of anemia. Consistent with reports in humans with fatigue syndromes, radiation‐induced CTRF correlated with increased levels of TNF and IL‐6. Additional work is ongoing to further characterize the model. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A28.