ICBG Project: Funded by the National Institutes of Health in cooperation with the Fogary International Center and NSF

International Cooperative Biodiversity Groups: Drug Discovery & Biodiversity Conservation in Madagascar - Planning Grant October 2003-September 2005

Progress Report, February 2005

Achievements in Biodiversity Conservation

Promoted Biodiversity Conservation: The ICBG program employed a team of local villagers who have prepared to collect plant samples once the signed contracts are approved by NIH. While the partner institutions have been negotiating these contracts, and until the contracts got final approval from NIH, the team has been collecting quantitative data on fruit and leaf phenology, habitat structure, and calculating population estimates for the trees at six sites throughout Ranomafana National Park. These data are currently being entered into the Park's long-term database and will be used for research, management, and estimating population densities used for medicinal plants.

Established Effective Working Relationships: The ICBG team has established close collaboration among all partners. In particular, a very efficient cooperative system between University of Antananarivo (LaCASN), IMRA, INDENA and Appendino-ICB&DD evolved. Bakolinirina Andriamihaja, chemist at the University of Antananarivo, has strengthened her close collaboration with Philippe Rasoanaivo, University of Antananarivo chemist and member of the Malagasy Institute for Applied Research (IMRA), a private Malagasy company dedicated to studying and developing medicinal plants in Madagascar. A December 2003 workshop at the Centre ValBio (at Ranomafana National Park) brought team members to the field site to develop plans and protocols for the project. Video conferences in early 2004 between Stony Brook University, Giovanni Appendino and INDENA allowed the team to develop extraction protocols. In May 2004, National Coordinator Bakolinirina Andriamihaja and IMRA Director Philippe Rasoanaivo visited the facilities at INDENA and Appendino's lab, in Italy. In June 2004, representatives of INDENA traveled to Madagascar to meet with Drs. Andriamihaja and Rasoanaivo, and visit the new laboratory built at the University of Antananarivo to support this project. Frequent email contact among all partners has allowed for the sharing of ideas and methodologies. These close collaborations provided the opportunity to test the project's chemistry protocol by using existing agreements to send commercially available plant materials to the Italian partners for assays. Monthly meetings between local villagers and the ICBG team, at the Centre ValBio, have encouraged interest in, dialogue about, and local support for the project.

Negotiated Agreements: The Research Foundation of Stony Brook University negotiated separate contracts with University of Antananarivo, the University of Fianarantsoa, and a joint contract with the Association of Village Leaders (Ampanjaka) and the Association of Traditional Healers (FIMARA) around Ranomafana. A joint Memorandum of Understanding was negotiated between the California Academy of Sciences, Research Foundation, and INDENA, SpA.

Structure of agreements among partners in the project

Every effort was made to ensure that negotiations were based on informed consent for all parties, as outlined in the ICBG "Principles for Access, Intellectual Property and Benefit-Sharing." Although Madagascar has not yet passed laws on IP and benefit sharing, we included language carefully defining IP and outlining benefit sharing for the Malagasy partners, using ICBG "Principles for Access, Intellectual Property and Benefit-Sharing." Our ICBG team worked with PIIPA (Public Interest Intellectual Property Advisors, Inc.) who provided us with the name of an attorney (Robert J. L. Lettington, Southern Environmental and Agricultural Policy Research Institute, based in Kenya) with expertise in IP and benefit sharing in Africa. Mr. Lettington spent a week in Madagascar (March 2004) advising ICBG staff, attorneys for the villagers, and attorneys for the Malagasy Universities on IP and benefit sharing issues. After negotiations that included the attorneys for the Malagasy partners, the project established the following benefit sharing scheme: should project discoveries result in royalty income, the parties agreed that the net royalty income would be split 50:50 between the three Malagasy Partners (University of Antananarivo, University of Fianarantsoa and the villagers around Ranomafana, as represented by FIMARA and Ampanjaka) and the three International Partners (ICB&DD, ICTE, and CAS). The villagers, University of Antananarivo, and University of Fianarantsoa met (July 2004) and signed a Convention to share the Malagasy portion of the net royalty income: 38% to Villagers, 31% each to the Universities. The villagers will receive their royalties or other income into a trust fund established in Madagascar for them at the time that royalties become available. The management of the trust fund is detailed in the contract. The international partners will share any royalties as outlined in the Memorandum of Understanding.

Ampanjaka and FIMARA pose for group photo in front of the Centre ValBio after signing the ICBG agreement

Define Economic Benefits with Villagers: The ICBG team works closely with two village organizations: the Ampanjakas (Association of Village Leaders) and FIMARA (Association of Traditional Healers). The ICBG team at Ranomafana National Park has held monthly meetings with village leaders to keep the team and the villagers informed about project activities, to develop economic activities to provide economic benefits to villagers from drug discovery, and to negotiate the contracts. The villagers and the ICBG team have developed a plan to create gardens/nurseries in villages and on farms to sustainably grow medicinal plants that are currently collected from the forest. A comprehensive "terms of reference (TOR)" has been written and a Malagasy consultant has been hired to develop a business plan to assist the villagers as they grow and market their medicinal plants. The request for a business plan came to the ICBG team directly from the villagers.

Achievements in the Pilot Project for Drug Discovery

Establishing efficient material transfer between Associate Programs. Access to materials is of great importance in drug discovery involving natural products. The multidiscipline-driven ICBG project requires the shipping of plants/extracts to concerned international partners. Material transfer is often a complicated matter in Madagascar in terms of administrative formalities, and as such we needed a simple and rapid way to deal with the matter while respecting the national regulations. To this end, we took advantage of the official partnership between IMRA and INDENA to set up and implement a material transfer arrangement. This arrangement has proved to be very successful in plant material transfer between Madagascar and Italy. Thus, this protocol will be continued and its possible expansion to the U.S. will be pursued in the Comprehensive Grant phase of this ICBG program.

Validation of extraction/pre-fractionation protocol and integrated screening system. Currently, methanol or ethanol is the most commonly used solvent to extract plants for biological screening, and the extract is generally submitted to liquid-liquid partition in order to minimize false positive/negative tests. However, methanol has been found to be a very poor solvent for most triterpenoids and aliphatic compounds, and has an opposite bias toward hydrophilic compounds. Furthermore, liquid-liquid partition may give strong emulsions. Dr. Appendino, Universitá del Piemonte Orientale (UPO) (Associate Program 4 Co-Leader) has devised an extraction/pre-fractionation protocol using acetone as an alternative solvent for extraction, followed by a rough silica gel column chromatography using respectively hexane, ethyl acetate and acetone as eluents. This technique was applied with great success in Madagascar (LaCASN and IMRA), first, in the case of Uapaca bojeri (Euphorbiaceae): While the crude acetone extract is completely inactive, the antiplasmodial activity is selectively concentrated in the ethyl acetate fraction (first elution). The UPO protocol appears to be efficient in identifying minor bioactive constituents. We then selected Malagasy plants with good ethnobotanical background, that were easily obtained via established trade, that came from genera with known bioactivity, had paucity or obsolescence of previous investigations [Dr. Rasanoivo (IMRA), Dr. Appendino (UPO), Dr. Almeda (CAS), and Dr. Wright (ICTE) contributed to the selection process]. The three species selected were: Cinnamosma macrocarpa (Canellaceae), Helichrysum cordifolium (Asteraceae), Harungana madagascariensis (Clusiaceae). Extracts and fractions of Cinnamosma macrocarpa were tested at IMRA for antiplasmodial and cytotoxic activities. Ethyl acetate fraction of Cinnamosma macrocarpa showed good in vitro antiplasmodial and cytotoxic activity (~1 µg/ml). Samples of the three plants were also shipped to an ICB&DD-affiliated Dr. Appendino's laboratory at UPO, based on the material transfer arrangement mentioned above. Different plant parts were extracted separately at UPO, and the pre-fractionated extracts assayed for cytotoxicity and apoptotic activities, inhibition of the nuclear transcription factor, NF-kB (a key player for inflammation and cancer), anti-Tat activity, anti-HIV-1 replication. Promising results were obtained. In particular, the cytotoxic activity of C. macrocarpa was confirmed (assays were performed at an ICB&DD-affiliated Dr. Muñoz's laboratory at the University of Córdoba (UCO), Spain), and the active fraction was further tested positively for apoptosis. Furthermore, Helichrysum spp gave the best results in terms of anti-inflammatory activities. This prompted the Madagascar team to buy Helichrysum cordifolium, which is widely used to treat inflammation, and which is sold in the market. This traditional use was confirmed by the NF-kB tests at UCO. The extracts also showed strong inhibition of the HIV replication at 50 µg/ml.

As exemplified by this "Pilot Project", we have confirmed that the exchange of knowledge and expertise between Associate Program Leaders is extremely beneficial for the project and we have successfully established such cooperative and productive close relationships among the Associate Programs. These preliminary results clearly indicate the validity of the extraction/pre-fractionation protocol and the integrated screening system that we implemented. It is highly likely that attractive hits/leads will be discovered from the unique biodiversity of Madagascar based on our validated approaches. It is worth emphasizing here that this Pilot Project has demonstrated convincingly that our Madagascar ICBG team has the required infrastructure and competence to conduct drug discovery research involving natural products locally in Madagascar by keeping close contact with the Group Leader and other Assoicate Program Leaders in a highly cooperative manner.

Development of Infrastructure in Madagascar

We have created the Laboratoire de Chimie Appliquée aux Substances Naturelles (LaCASN) at the University of Antananarivo, under the direction of Dr. Bakolinirina Andriamihaja (National Coordinator of the ICBG Planning Grant in Madagascar). We have funded renovation of the space, and the purchase of benches, hoods, equipment, and office materials needed for plant extractions and fractionations. In addition, we have provided supplies and equipment to the ISTE laboratory at the University of Fianarantsoa. The LaCASN is already under full operation for the extractions of medicinal plants. These laboratories will play a crucial role in the extractions and bioactivity-guided (pre)fractionations of medicinal plants and lichens. At the Centre ValBio near Ranomafana National Park, teams of villagers have been hired and trained to collect plant samples after the contracts have been approved by NIH. LaCASN, CAS, and ICTE team have collaborated closely with Dr. Rasoanaivo (IMRA) to develop protocols for material and data exchange and have developed a database for uniform coding of plants and extracts. California Academy of Sciences has constructed a walk-in plant drier at the Centre ValBio for use by the project.