Eburnamonine Research Articles

Investigating the stability of a cerebral vasodilator drug using chromatographic methods: Evaluation of methods’ practicality and environmental aspects

A vinca alkaloid; vinburnine (VNB) is utilized as an effective vasodilator. As a cyclic amide-containing drug, it is likely susceptible to hydrolytic degradation. This study examined the degradation profile of VNB, findings indicated that VNB undergoes degradation solely in the presence of alkali, generating a carboxylic acid derivative (DEG). The present study aimed to design and apply green TLC-densitometric and RP-HPLC assays for concurrently measuring VNB and its degradation product for the first time. TLC-densitometric assay was carried out on silica gel 60 F254 TLC plates and a developing system of ethyl acetate: methanol: triethylamine (6:4:0.05, by volume) and detection at 230 nm. RP-HPLC method depended on a C8 column and a mixture of methanol: water (95:5, v/v). The rate of flow was 1 mL/min and UV detection at 230 nm. The proposed assays were used for prediction of the degradation behavior of VNB under the mentioned conditions and then applied for quantitation of VNB in its commercially available capsules. Four distinct metric approaches; National Environmental Method Index (NEMI), Analytical Eco-Scale, Green Analytical Procedure Index (GAPI), and Blue Applicability Grade Index (BAGI) were utilized to assess the chromatographic method’s ecological effect. Findings obtained from the provided methodologies were contrasted statistically with the stated HPLC method using Student’s t and F-tests. The analysis revealed that there were no significant differences between them. The established methods were verified in accordance with the recommendations of the International Council for Harmonization (ICH), and all the outcomes were deemed to fall within the permissible limit.

Evaluation of vinburnine in pharmaceuticals by smart spectrophotometric methods; full stability study

Vinburnine (VNB) is a vinca alkaloid used as a vasodilator to enhance cerebral circulatory insufficiency. It is a cyclic amide containing drug which is expected to be sensitive to hydrolytic degradation. The degradation profile of VNB was studied in this work following ICH recommendations for stability study. The drug was sensitive only to degradation with NaOH with the formation of the carboxylic acid derivative, identified by IR and 1H NMR analyses as 2-((1S,12bS)-1-ethyl-1,2,3,4,6,7,12,12b-octahydroindolo[2,3-a] quinolizin-1-yl) acetic acid, (DEG). In this study five simple, smart and univariate stability indicating spectrophotometric methods were developed and validated for simultaneous determination of VNB and DEG for the first time. The developed methods include; Dual Wavelength Method (DWM), Dual Wavelength Resolution Method (DWRM), Factorized Absorbance Difference Method (FADM), Advanced Absorbance Subtraction Method (AASM), and Derivative Amplitude Factor Method (DAFM). These methods were capable of determination of VNB and DEG over the ranges of 1–30 and 3–50 µg/mL, respectively. The proposed methods were simple, smart, specific, and could be applied for analyzing synthetic mixtures of VNB and DEG and were successfully applied for determination of the drug in commercially available capsules. The obtained results of these methods were statistically compared with the reported HPLC one using student's-t and F- tests, where no significant difference was observed. Validation of the developed methods was applied according to ICH recommendations and all the results were within the acceptable limits.

Abstract B30: 15-Methylene-Eburnamonine: A preventative agent against breast to brain metastases by targeting circulating tumor cells and cancer stem cells through induction of oxidative stress

Abstract One in eight women in the United States will develop invasive breast cancer during their lifetime. Of these women, a significant portion (10-16%) will develop metastases to the brain, resulting in significant morbidity and poor prognosis. Symptoms of brain metastasis are devastating and include dizziness, nausea, vertigo, headaches, impaired vision and cognitive functions. Brain metastases arise from highly aggressive triple negative (estrogen, progesterone, and Her2 receptor negative) or Her2 positive (estrogen and progesterone receptor negative, and Her2 positive) mammary tumors and often afflict young, premenopausal women. Less than 2% of women with such lesions survive past 2 years. Since there are currently no effective therapies against brain metastases, the best approach is to prevent their formation by targeting circulating tumor cells before they engraft at distant sites. It has been suggested that cancer stem cells (CSCs) contribute to the metastatic spread in many solid tumors, including those of the breast. A successful preventative agent must kill circulating tumor cells and CSCs before they have a chance to colonize a secondary site while having minimal toxicity against normal tissue. Eburnamonine (EBN) is a pharmacologically active compound derived from the flowering plant Periwinkle with vasoregulatory and anti-hypoxic properties. It has been found to cross the blood-brain-barrier and has been used as a treatment for a number of cerebrovascular disorders, including ischemia and anoxia. EBN has no known neurotoxicity, in fact it has been found to be neuroprotective. A derivative of EBN, 15-methylene-(-)-eburnamonine (15-M-EBN), with increased cytotoxity against cancer cells has been synthesized by our group. We found that EBN is cytotoxic against triple negative human breast cancer cells, MDA-MB-231 (LC50=42.0 μM). Our derivative, 15-M-EBN, is even more effective against triple negative human breast cancer cells, MDA-MB-231 (LC50=14.0 μM), as well as against triple negative breast to brain metastatic cells, MDA-MB-231BR (LC50=26.4 μM); and Her2 positive breast to brain metastatic cells, MDA-MB-231BR-Her2 (LC50=27.2 μM). Mammosphere assay indicated that 15-M-EBN kills CSCs found in MDA-MB-231, triple negative breast cancer cells (LC50=10.3 μM), as well as those found in triple negative breast to brain metastatic cells, MDA-MB-231BR, (LC50=26.2 μM) and Her2 positive breast to brain metastatic cells, MDA-MB-231BR-Her2 (LC50=19.8 μM). Also, by using 3D cultures and coatings of various extracellular matrix component proteins, we established that 15-M-EBN is active under the conditions of environmental-mediated drug-resistance. Staining with Annexin V showed that 15-M-EBN induces apoptosis that we demonstrate to be mediated through the activation of caspases 3, 6, and 7 and cleavage of cytokeratin 18. Addition of reducing agents (N-Acetyl-L-cysteine, glutathione, or DTT) to 15-M-EBN treated cultures blocked its cytotoxic effects, demonstrating that 15-M-EBN acts through the induction of oxidative stress. Furthermore, to establish that the methylene group on the fifteenth carbon in the structure of 15-M-EBN is the reactive site, our group synthesized a compound with a methyl group in place of the methylene (15-methyl-EBN). It was tested in cultures of breast cancer cells and showed that replacing the methylene with the methyl group abrogates the cytotoxic effects of this compound. Taken together our data show that 15-M-EBN kills triple negative and Her2 positive breast cancer cells and CSCs derived from these tumors through an induction of oxidative stress, overcomes the pro-tumorigenic effects of the tumor microenvironment while exhibiting minimal toxicity against normal tissue. These findings suggest that 15-M-EBN has a high potential to become a preventative agent against breast to brain metastases. Citation Format: Mary Minyi Zheng, Britney Harris, James Woods, David Colby, Julia Kirshner. 15-Methylene-Eburnamonine: A preventative agent against breast to brain metastases by targeting circulating tumor cells and cancer stem cells through induction of oxidative stress. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr B30.

Abstract 1984: Nature's pharmacy: eburnamonine and its derivative as therapeutics against brain tumors

Abstract Brain tumors are highly aggressive and often afflict younger patients. In 2011 there were 22,340 estimated new cases of brain tumors in the United States with 40% arising from metastatic lesions originating mostly from lung, breast, and melanoma tumors. Treatment options remain limited and include steroids, whole brain radiation, and surgery. Because of difficulty penetrating the blood-brain barrier (BBB), chemotherapy is usually not a viable option. Therefore, any intervention that can block the malignant colonization of the brain and/or eliminate symptomatic and occult brain lesions will significantly improve both quality of life and survival. To target brain tumors, a successful therapy has to meet the following criteria: 1) penetrate the BBB; 2) exhibit specificity for the tumor cells; and 3) have minimal off-target effects. Here we investigate a Periwinkle minor derived natural product, eburnamonine (EBN), and its structural analog, 15-methylene-(-)-eburnamonine (15-M-EBN), as therapeutic agents against brain lesions from glioblastoma multiforme and metastatic breast cancer. The two common subtypes of breast tumors giving rise to brain metastases are triple negative and Her2 positive primary tumors. Potency of EBN and 15-M-EBN was evaluated in cultures of chemosensitive (U87) and chemoresistant (SF767) glioma cell lines and ‘brain-seeking’ metastatic breast cancer cells, MDA-MB-231BR (triple negative) and MDA-MB-231BR-Her2 (Her2 positive). To test whether our compounds can overcome the environment-mediated drug resistance (EM-DR) cells were treated in the context of extracellular matrix (ECM) (hyaluronic acid, collagen IV/laminin, and collagens I and IV/laminin/fibronectin to reconstruct the brain, mammary gland, and bone marrow microenvironments respectively). The off-target toxicity of EBN and 15-M-EBN was tested on normal neuronal cells, bone marrow stromal cells, and non-malignant mammary epithelial cells. To evaluate the capacity of EBN and 15-M-EBN to penetrate the BBB, we utilized a transwell model with hCMEC/D3 cell monolayer mimicking the BBB. We demonstrate that EBN and 15-M-EBN are cytotoxic against glioma and ‘brain-seeking’ metastatic breast cancer cells (LC50=42 micromolar and 14 micromolar respectively) with minimal toxicity against non-malignant cells. Moreover, 15-M-EBN is active in the tumor environment and predicted to cross the BBB and have anti-cancer stem cell activity. Our preclinical data establish that 15-M-EBN meets the major criteria for a successful therapeutic agent to target brain lesions, both primary and metastatic, due to its ability to: 1) exhibit specificity for the tumor cells; 2) have minimal neurotoxicity and off-target effects; and 3) penetrate the BBB. In vivo studies are in progress to establish a pro-drug strategy to increase water solubility and bioavailability of 15-M-EBN for further clinical development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1984. doi:1538-7445.AM2012-1984

Effects of vinburnine on experimental models of learning and memory impairments

Retrograde amnesia can be induced experimentally in mice injecting them with scopolamine (3 mg/kg, IP) or by inducing seizures with pentylenetetrazol (50 mg/kg, IP), and in rats by subjecting them to hypobaric hypoxia (at a barometric pressure of 300 mmHg for 3 min). We have studied the effects of vinburnine (VNB) in these amnesic states compared to vincamine (VNC) and nicergoline (NCG), in order to assess its activity on drug-induced learning and memory impairments. Vinburnine reduced the disrupting effect of both scopolamine and pentylenetetrazol-induced seizures on the retention of a step-through passive avoidance behavior in mice and on the acquisition of shuttle-box active avoidance behavior in rats. This effect was dose-related up to 20 mg/kg, the peak effect dose after IP administration, and more pronounced than that of VNC and NCG in some tests. These results indicate that VNB influences learning and memory processes disrupted by a pharmacological manipulation. In particular, as scopolamine acts as anticholinergic drug, it is possible that VNB mechanism of action includes also a stimulation of acetylcholine neurotransmission.

ChemInform Abstract: Synthesis of Eburnamonine and Dehydroaspidospermidine.

Syntheses a partir d'acetyl-5 tetrahydro-1,2,3,4 pyridine via l'ethyl-3a perhydro furo [2,3-b] pyridinone-2

Synthesis of eburnamonine and dehydroaspidospermidine

Syntheses a partir d'acetyl-5 tetrahydro-1,2,3,4 pyridine via l'ethyl-3a perhydro furo [2,3-b] pyridinone-2

The Alkaloids of Hunteria eburnea. I. The Structures of Eburnamine, Isoburnamine, Eburnamenine and Eburnamonine and a Synthesis of rac-Eburnamonine1

The Alkaloids of Hunteria eburnea. I. The Structures of Eburnamine, Isoburnamine, Eburnamenine and Eburnamonine and a Synthesis of rac-Eburnamonine¹