The Effect of Antineoplastons A10 and AS2-1 and Metabolites of Sodium Phenylbutyrate on Gene Expression in Glioblastoma Multiforme

Antineoplastons are peptide and amino acid derivatives that occur naturally in the human body. They inhibit the growth of neoplastic cells without growth inhibition of normal cells. Phenylacetylglutaminate (PG) is an active ingredient of antineoplastons A10 and AS2-1 (ANP) and is also a metabolic by-product of phenylbutyrate (PB). The formulation of antineoplaston AS2-1 is a 4:1 mixture of phenylacetate (PN) and PG. Antineoplaston A10 is a 4:1 mixture of PG and isoPG. This study investigates the molecular mechanism of action of PG and PN. The Human U87 glioblastoma (GBM) cell line was used as the model system in this study. A total human gene array screen using the Affymetrix Human Genome plus 2.0 oligonucleotide arrays was performed using mRNA derived from U87 cells exposed to PG and PN. Pathway analysis was performed to allow the visualization of effect on metabolic pathways and gene interaction networks. Our preliminary results indicate that PG and PN interrupt signal transduction in RAS/MAPK/ERK and PI3K/AKT/PTEN pathways, interfere with cell cycle, decrease metabolism and promote apoptosis in human U87 GBM cells. The effect on multiple cellular pathways and targets, suggests that ANP and PB are promising candidates for clinical studies in GBM.

Preliminary Findings on the Use of Targeted Therapy in Combination with Sodium Phenylbutyrate in Colorectal Cancer after Failure of Second-Line Therapy—A Potential Strategy for Improved Survival

Colorectal cancer (CRC) is the second leading cause of cancer death related mortality with 1.2 million new cases diagnosed annually worldwide. Despite remarkable advances in the treatment of resectable CRC, advanced disease that recurs following initial two lines of chemotherapy, remains incurable. Targeted therapies using a single agent or in combination with other drugs have been tested in a number of clinical trials, with only moderate improvement. Here we present preliminary findings of improved overall survival (OS) using a combination of sodium phenylbutyrate with various targeted and chemotherapeutic agents in stage IV CRC patients who had failed at least two lines of chemotherapy. Results suggest a strategy of simultaneous interruption of signal transduction involving EGFR (VEGF)?KRAS-ERK and PI3K-AKT pathways and interference with cell cycle, cancer cell metabolism, maintenance of cancerous stem cells, and promotion of apoptosis. In a group of 15 patients, median OS was higher compared to other third-line therapies (14.7 months compared to between 4.8 and 9.5 months in other studies). Given the understanding that our findings are preliminary, we propose the validation of our initial results using a well-designed phase I/II trial in recurrent advanced colorectal cancer.

GENE EXPRESSION PROFILING OF PHENYLBUTYRATE INDUCED DIFFERENTIATION OF GLIOMA CELLS BY cDNA ARRAY

Objective: To analyze the changes of gene expression in phenylbutyrate induced differentiation of glioma cells. Methods: The expression levels of 14000 genes in glioma cells before and after inducement with sodium phenyl- butyrate for 2 h or 6 days were evaluated by cDNA array technique and proved by multi-dot blotting. Results: expression of 98 genes in glioma cells showed changes after the inducement. Some genes involved in transcription and translation and some oncogenes are down-regulated, while some gene involved in differentiation or apoptosis are up-regulated. 18 unknown expression sequencing tag (EST) changed too. Conclusion: A gene expression profile associated with differentiation of glioma cells was established.

Preliminary Findings on the Use of Targeted Therapy in Combination with Sodium Phenylbutyrate in Recurrent Advanced Pancreatic Cancer—A Potential Strategy for Improved Survival

Metastatic pancreatic cancer carries an estimated five-year survival rate of only 2%. Gemcitabine-based chemotherapy remains a first-line standard-of-care treatment for elderly patients with advanced pancreatic cancer. Combination chemotherapy FOLFIRINOX offers better results, but it is not recommended for the older patient population due to substantial toxicity. Standard-of-care second-line treatment is not yet established and is used in approximately 30% of patients since performance status is too low to consider further therapy. Targeted therapies with a single agent and in combinations have been tested in numerous clinical trials, but except for the combination of gemcitabine and erlotinib, have not yet proven efficacy. Here, we present preliminary findings of improved overall survival (OS) using a combination of sodium phenylbutyrate with various chemotherapeutic and targeted agents in stage IV A and B pancreatic cancer patients who failed at least one line of chemotherapy. The results suggest a strategy of simultaneous interruption of signal transmission involving multiple pathways in the second-line treatment that are believed to interfere with cell cycle, cancer cell metabolism, autophagy and maintenance of cancer stem cells and promote apoptosis. In this group of patients, median OS was higher compared to other second-line therapies (10.5 months compared to between 2.9 and 6.5 months in other studies, and in the best supportive care group, 2.3 months). Given the understanding that our findings are preliminary, we propose the validation of our initial results using a well-designed Phase I/II trial in recurrent advanced pancreatic cancer.

Preliminary Findings on the Use of Targeted Therapy with Pazopanib and Other Agents in Combination with Sodium Phenylbutyrate in the Treatment of Glioblastoma Multiforme

The most common and aggressive type of brain tumor is glioblastoma multiforme (GBM). The prognosis for GBM remains poor with a five-year survival rate between 1% and 2%. The prospects for patients with recurrent GBM (RGBM) are much worse, with the majority dying within 6 months. This publication provides a brief description of the treatment of 11 GBM patients treated with sodium phenylbutyrate (PB) in combination with pazopanib, m-TOR inhibitors, and other agents. The treatment was associated with tolerable side effects and resulted in objective responses in 54.5% of cases (complete response 18.2%, partial response 36.3%) and 27.3% cases of stable disease. The preferable treatment regimen consisted of PB, pazopanib, dasatinib, everolimus, and bevacizumab (BVZ). For various reasons not all patients were compliant with the treatment regimen. In patients who strictly complied with the treatment plan, all responded as CR or PR. Based on preliminary findings, the authors propose further phase I/II clinical trials with PB in combination with pazopanib, dasatinib, everolimus, and BVZ in patients with RGBM who failed standard surgery, radiation therapy and chemotherapy. With proper dose reductions, the treatment appears to be well-tolerated. Molecular profiling of patient subgroups with favorable genomic signatures may help to select patients for future studies.

A Case of Sustained Objective Response of Recurrent/Progressive Diffuse Intrinsic Pontine Glioma with Phenylbutyrate and Targeted Agents

Diffuse intrinsic pontine glioma (DIPG) is the most common type of brainstem glioma and one of the most deadly brain tumors. DIPG in young adult patients is a rare disease for which treatment options are limited. Radiation therapy remains the standard-of-care for newly-diagnosed DIPG, but no established therapies for recurrent disease are available. This paper describes the results of treatment of a young adult patient diagnosed with DIPG that progressed after radiation therapy. Therapy included sodium phenylbutyrate (PB) in combination with the targeted agents: pazopanib, everolimus, erlotinib, and bevacizumab. The patient achieved a rapid partial response, which persisted over a year and five months. The patient opted to discontinue the therapy and thereafter elected chemotherapy, which resulted in a subsequent rapid progression and death within one month. The targeted treatment was associated with minor toxicity that included a Grade 2 skin rash and Grade 1 elevation of transaminases. In conclusion, a combination of PB and currently available targeted drugs may offer extended survival in patients with recurrent DIPG.

Treatment of Esthesioneuroblastoma and Nonsmall Cell Lung Cancer with Phenylbutyrate

Esthesioneuroblastoma is a malignant tumor, arising in the upper nasal cavity, that could spread to the frontal lobe of the brain as well as metastasize to the lymph nodes. Due to the low incidence of this tumor, FDA-approved treatment modalities do not exist and clinical trials have not been performed. We present an interesting case of a 66-year-old female, diagnosed with Kadish stage B esthesioneuroblastoma and stage IIA nonsmall cell carcinoma of the lung, who benefited from our treatment. Both malignancies were diagnosed in 2002 at which time the patient consented to undergo left upper lobectomy for her lung cancer, but she refused the craniofacial resection and radiation therapy recommended for treatment of her esthesioneuroblastoma. From 2003 to 2004 she received treatment at the Burzynski Clinic with oral sodium phenylbutyrate (0.2 g/kg/day). She tolerated the treatment very well without significant adverse events. Gradual reduction in her tumor size was confirmed by repeat MRIs. From treatment start in March 2003 to December 2003 her tumor decreased by 40%. Subsequent MRI from March 2004 revealed increased tumor size, which, however, was still a 13% reduction from the baseline MRI. What is important to mention is that in addition to shrinkage of the esthesioneuroblastoma, the patient obtained the clinical benefit of 3.5-years longer survival than was predicted for her lung cancer—whereas the median survival for a patient with stage IIA adenocarcinoma of the left upper lobe of the lung is approximately two years, our patient survived more than five and a half years. The effect of phenylbutyrate (PB) and its metabolite phenylacetate on neuroblastoma and lung cancer is documented by numerous preclinical studies and is also evident in this case. It is proposed that the activity of these two compounds is mediated through increased expression of the p21 tumor suppressor gene. p21 is a strong inhibitor of cyclin-D and cyclin-dependent kinase 4, which contribute to undifferentiated phenotype in neuroblastoma and are instrumental in cell cycle progression from G1 to S phase. It is hoped that future research and combination of PB with other chemotherapeutic and targeted agents will provide better control of esthesioneuroblastoma and lung cancer.

Preliminary Findings on the Use of Targeted Therapy in Combination with Sodium Phenylbutyrate in Advanced Malignant Mesothelioma: A Strategy for Improved Survival

Advanced malignant mesothelioma (MM) is among the most aggressive and difficult-to-treat diseases. Industrialization and exposure to asbestos is the main causative factor for the dramatic increase in the incidence of MM, which carries a poor prognosis and a median survival of less than 12 months. Combination chemotherapy offers only palliative results;however, targeted therapy carries more promise for future successful treatment. This paper presents preliminary findings of improved overall survival (OS) using a combination of sodium phenylbutyrate (PB) with various chemotherapeutic and targeted agents in advanced MM. The data suggest using a strategy of simultaneous interruption of signal transduction involving RAS-MEK-ERK, PI3K-AKT, mTOR, Merlin, and angiogenesis pathways and interference in cell cycle and epigenetic processes. Complete response was determined in 15.4% and stable disease in 46.2% in the group of 13 evaluable patients. Median OS for MM was higher compared to other treatments (17 months compared to between 6 and 12.1 months). The longest surviving patient continues to be in complete response and in excellent condition for over 12.5 years from the treatment start. These findings are only preliminary and validation of the results using a well-designed phase I/II trial in advanced MM is proposed.

Recurrent Glioblastoma Multiforme—A Strategy for Long-Term Survival

Recurrent GBM (RGBM) has a highly unfavorable prognosis with majority of patients dying within 6 months and no standard treatments available. Antineoplaston (ANP) A10 and AS2-1 injections underwent Phase II trials in RGBM patients, which reported a long-term overall survival (OS) in a small percentage of patients. The additional Phase II studies BT-07, and BT-21 with ANP in GBM also revealed cases of a long-term OS. ANP shares active ingredients with metabolites of sodium phenylbutyrate (PB), which was used in private practice setting in combination of targeted and chemotherapeutic agents for the treatment of RGBM. The treatment contributed to cases of rapid complete response (CR) and significant OS. This paper provides case studies of three patients treated with ANP under Phase II protocols and two patients treated with PB in combination with targeted therapy, who obtained CR and long-term OS. Based on these studies and basic research on the effects of ANP and PB on the genome of GBM and review of results of preclinical and clinical research on targeted agents, the authors suggest a new strategy for successful treatment of RGBM. They propose Phase I/II clinical trials with ANP and PB in combination with targeted agents, bevacizumab (BVZ), pazopanib, dasatinib and everolimus in patients with RGBM after failure of standard surgery, radiation therapy (RT) and chemotherapy including temozolomide (TMZ) to be conducted to evaluate survival, response and toxicity in these patients.