Macrofil chemotherapy project

JOINT PROGRAMME COMMITTEE Office of the Chairman COMITE CONJOINT DU PROGRAMME Bureau du Prdsident Onchocerciasis Control Programme in West Africa Programme de lutte contre l'onchocercose en Afrique de l'Ouest African Programme for onchocerciasis Control Programme africain de lutte contre l'onchocercose JOINT ACTION FORUM Office of the President FORUM D'ACTION COMMUNE Bureau du Pr6sident JPCzz-JAF7IINF/DOC.I JPC - JAF CCP _ FAC JOINT SESSION / SESSION CONJOINTE Washington DC, U.S.A., 12 December 2001 MACROFIL CHEMOTHERAPY PROJECT t1 MACROFIL CHEMOTHERAPY PROJECT Goals The goal of MACROFIL (FILARIASES R&D) is to develop or optimize tools for the detection, evaluation and treatment of onchocercisais. 2. The primary emphasis is on discovery and development of a treatment that could be used in the context of the Onchocerciasis control programmes, and would result in permanent sterilization or killing of adult worrns. 3. The secondary emphasis is in discovering molecular indicators in Onchocerca volwlus that could be correlated with a non responsive or ivermectin resistant parasite. This knowledge will be used to develop a field tool for the control prograrnmes to detect ivermectin resistance if or when it may occur. In conjunction with this, targeted clinical observations/studies in the field are undertaken to asses if ivermectin resistance in patients is occurring or not. Clinical activities 4. Ivermecttn combinations for onchocerciasis: Dr. K. Awadzi of the Onchocerciasis Chemotherapy Research Centre (OCRC) concluded two key clinical studies: D The safety, pharmacokinetics, microfilaricidal and macrofilaricidal effect of co- administration of ivermectin and albendazole: The combinations were equipotent to ivermectin alone in their effect against the microfilariae; they did not offer advantage over ivermectin on the effects on the reproductive activity of the adult female worms or worn viability. The combinations of drugs were well tolerated and did not produce more severe adverse effects than ivermectin alone. No pharmacokinetic drug interactions were detected. ii) The safety and pharmacokinetics levamisole plus ivermectin and levamisole plus albendazol : As above, no advantage over ivermectin alone was detected. The results of these studies are being prepared for publication. 5. Moxidectin ; Moxidectin is a registered animal health product (American Home Products through American Cyanamid) for which the effect on experimental filariases animal models has been extensively studied by TDR sponsored scientists. Following informal consultations with American Home Products through its Wyeth-Ayerst Research Division the available data were considered adequate to justiff a drug development programme. A human clinical study in healthy normal (O. volvulus negative) volunteers in UK to evaluate safety, tolerability and pharmacokinetics of moxidectin has been initiated (ongoing). Based on the results from this study (January 2002) a decision to proceed with the clinical development of this product as a tool for onchocerciasis control will be discussed between the "Company", APOC, OCP, and TDR. The costs of the clinical studies will be covered through MACROFIL, while the commercial parhrer will be responsible for all aspects related to the pharmaceutical development and product registration. The end point that will be pursued by this development programme is the demonstration that treatment of Onchocerca infected individuals with single dose of moxidectin will result in longlasting disappearance of microfilaria from the skin (death or permanent sterilization of adult worms). This effect must be achieved with safety and tolerability comparable to or better than that of ivermectin. 2Wolbachia: long term use of antibiotics as potential chemotherapeutic agents against filariases. i) Doqtc:tcline.' A recent study from the Bernhard Nocht-Institute for Tropical Medicine, Hamburg, Germany, has shown that administration of doxycycline (daily 100 mg oral dose) for at least 6 weeks to Onchocerca volvulrzs infected patients (previously treated with ivermectin in Ghana), resulted in disappearance of skin microfilaria that lasted at least 18 months. It is assumed that this effect is associated with the antibiotic effect of doxycycline on Wolbachia, an endosymbiont of f,rlarial parasites. Based on this clinical observation, several clinical research proposals addressing further clinical examination of this antibiotic in onchocerca infected patients have been submitted to TDR. After a critical review it was decided not to endorse further clinical studies with doxycycline primarily because there was no indication that shorter treatment periods would be effective or that higher doses would be more effective. In addition even if proven effective, the long term use of doxycycline was seen as not compatible with the needs and limitations imposed by the nature of the onchocerciasis control prograrnmes. The contraindication of use during early pregnancy would impose pregnancy exclusion or prevention of pregnancy during the length of the treatment (6 weeks). Due to similar contraindication in children they would be excluded from the therapeutic benefit as well. Furthermore, long-term use of doxycycline could provide selective pressure towards drug resistance in life threatening microorganisms. On the other hand it was recognized that doxycycline could be considered for specific case management of individual onchocerciasis patients or in special communities. Based on these discussions it was acknowledged that Wolbachia represents a suitable target and it was recommended that the drug discovery group in TDR conduct appropriate studies to identi$r suitable drug candidates ' against Wolbachia (see below). iD Rifampicin ; Experimental observations in animals have shown that rifampicin, a commonly used drug in the treatment of tuberculosis and also as an antirickettsial agent, is effective against Wolbachia. To further validate the Wolbachia as a target for treatment of filariases we have promoted a human clinical study aimed to evaluate the brugian filariasis infection in patients with pulmonary tuberculosis who are culrently being included in the Directly Observed Treatment - Short course (DOTS) progralnme. These patients are receiving rifampicin for a period of six months. A case study of patients arriving for TB treatment at the tuberculosis unit in T.D.Medical College Hospital (departments of Internal Medicine, Chest and Tuberculosis, Paediatrics and the District T.B.Centre), in collaboration with the Filariasis Chemotherapy Unit at Allepey, India has been initiated. Patients are screened for microfilariae (night blood examination) before they receive DOTS (including rifampicin) and those who have microfilaria due to Brugia malayi infection will be evaluated at intervals of two, four and six months. The study will assess the long-term efficacy (reduction of blood microfilaria) of a therapy containing rifampicin without interfering with the tuberculosis treatment. 6. Human pharmacokinetic sa-fetv and e.fficac]t studies of albendazole + ivermectin / DEC combtnati'ons in ZF: Clinical studies relevant for the Global LF elimination programme and its integration within the Onchocerciasis control programmes were initiated and are ongoing: i) Albendazole *ivermectin: safety, efficacy in LF and intestinal helminthes study in Pemba Island (Dr. M. Dahoma). 1000 subjects including LF positive. The treatment phase of this study has been completed; currently the patients are being followed for the eff,rcacy and safety. The study is expected to be completed by December 2001. aJ ! ii) Albendazole * DEC: pharmacokinetics, safety study (Dr. K. Shenoy) This study demonstrated that there is no pharmacokinetic interaction between albendazole and DEC. iii) Albendazole+DEC: safety, efficacy and PK (Dr. N. Kshirsagar) 1400 subjects from a LF endemic region in India have been treated, the study results will be available December 2001. 7. Clinical stuht to evaluate individuals from selected OCP areas with skin microf,rlarial profiles not congruent with the expected results after repeated ivermectin treatments. 8. The epidemiological and entomological analysis of data from OCP highlighted that some individuals from the Bui-Black Volta and Pru (Ghana) areas, despite having received ivermectin 9 or more times, still had skin microfilarial loads above those that were expected (10 or more microfilariae/snip). From consultations between OCP, OCRC, Ghana National Onchocerciasis Control prograrnme, TDR and experts from the Noguchi Institute for Medical Research in Ghana it was decided to study these individuals. Under strict clinical observation (at OCRC) these individuals were treated with the recommended normal dose of ivermectin. To determine if the drug was appropriately absorbed and metabolised, drug blood levels were determined. In addition the drug effect on the parasites was also determined by quantifoing skin microfilariae before and after treatment as well as by quantiffing and evaluating microfilarial survival in black flies. Furtherrnore, ocular examination and nodule ultrasonography and histopathology to determine the viability and fertility of adult worrns will be measured. The isolated parasites from these patients will be examined by the team of molecular biologists who will look for changes in key genes. The study was initiated in April 2001 and results are expected for July 2002. Pre-clinical research activities Ivermectin resistance detection tool 9. The objective of this project is to develop a PCR-based assay to detect ivermectin resistance by assessing shifts in the allele frequencies at loci that may serve as markers for ivermectin resistance in O.volvulzs. Two focused teams are collaborating, one in West Africa undertaking fieldwork (see above clinical work) and one international undertaking laboratory studies. During the first year of the project the identification of candidate genes associated with ivermectin resistance in Haemonchus contortus and C. elegans was completed. 10. In the second year (reporting year), candidate O. volvulus genes from adult and microfilaria material from "unselected" (ivermectin untreated areas) and ivermectin exposed areas were examined for polymorphism. "Association analysis" of selected markers from these samples were conducted. In addition a DNA preparation method compatible with the present method of sample collection during routine field activities (skin snips or Black flies) was developed in conjunction with the OCP Laboratory team in Ouagadougou. 11. During the third year (2002), genes showing polymorphism will be evaluated for development of a PCR assay to detect the presence of alleles more frequent in ivermectin selected areas. At the end of the third year a "proof of concept" PCR assay to detect allelic changes in O. volvulus candidate genes will be completed. il 4Optimisation of DN slcanslehle;! 12. Unfortunately, this project has been on hold because the negotiations regarding the development of a standardized product based on a transdermal drug delivery technology from a German company have not progressed as expected. Recent direct conversations between Dr. B. Liese and the management of this company have brought the potential partners back on the negotiation table and the R & D activities for prototype production have been initiated.. Drug discovery activities Inhibitors apainst lilarisl snd Wolbachia specilic LRNA synthetases 13. Structure-based drug design has revolutionized the development of new therapeutics for microbial infections, HIV and cancer. This provides a unique opportunity for a university-based collaboration lead by Dr. Michael Kron (Michigan State University) to design and test inhibitors of a filarial parasite enzyme known as an aminoacyl-tRNA synthetase (AARS). This enzyme is a novel validated molecular target for anti-infective drugs discovery, recognized by those in the pharmaceutical industry responsible for development of new antibacterial and antifungal drugs. The team is applying four new technologies to identiff inhibitors of an AARS from the human parasite: a Production of recombinant enzyme and purification on a large scale. The enzymes have proven stable enough for use in robotic "high throughput screening" formats, in which enzyme inhibition is measured after exposure to each of thousands of different potential chemical inhibitors. Use of a drug design methodology called SLIDE (Screening for Ligands with Induced-Fit Docking, Efficiently ) developed at Michigan State University has previously been applied only in the private sector pharmaceutical industry. This method performs computer based screening of chemical databases and predicts the best candidate enzyme inhibitor by attempts to "dock" them into the 3-dimensional structure of the protein target. Successful determination of the three dimensional atomic structure of a filarial AARS, through partnership with Dr. Stephen Cusack, Director of the European Molecular Biology Laboratory Grenoble Outstation crystallography group. This is the first such enzyme structure ever solved. Collaboration with synthetic organic chemists committed to drug design (Oslo, Norway and Christchurch, New Zealand) providing the opportunity to synthesize and optimize the molecular "scaffolds" identified by other methods. Synthesized or natural compounds identified by different screening methods are tested again in vitro to see if they inhibit recombinant eruyme or in vivo for activity against adult worms maintained in culture. These steps provide feedback to refine each phase of the inhibitor design cycle. 14. Approximately 100,000 potential inhibitors of the filarial erzyme have been analyzed, and we have secured the methods to continue analysis of up to 400,000 more. The research has identified 17 new chemical structures (synthetic and natural products) that inhibit parasite AARS, as lead antifilarial compounds for further study. The successful implementation of our drug discovery strategy has drawn the attention of an international pharmaceutical company, Basilea Inc. (the anti- infective company spun-off from Roche), that has pledged its future support by providing medical chemistry advice as well as access to its private collection of novel chemical libraries. 15. Similarly, to find lead compounds to klllWolbachia,the teamhas cloned and are expressing a very unique AARS from the Wolbachia living within adult female Brugia malayi parasites. After 1 a a I r] 5I purification of this enzyme the above described strategy will be followed: - determination of the Wolbachia AARS structure, use of SLIDE and high throughput screening. 16. Thus, by careful selection of an interdisciplinary and multinational research team, a unique molecular target and incorporation of new technologies of great interest to private sector goals for "profitable disease drug discovsr/," we are making important strides toward rational development of anti-filarial and anti-llolbachia drugs. Whole parasite drug screening in in vitro or in vivo. 17. Substances from available chemical libraries as well as specific substances (antibiotics) are examined in the Laboratory of S. Townson. Compounds are first examined in in vitro 5 day assay against male Onchocerca gutturosa.. This has been useful in identiffing 5 antibiotics (rifampicin, minocycline, doxycycline, clofazamine and ethambutol) with significant anti-Onchocerca activity. Active compounds are further tested in vivo against Onchocerca microfilariae in mice and against transplanted Brugta pahangi macrofilariae in jirds. Positive compounds are further evaluated in Brugia pahangi in dogs. 18. While a substantial number of known drugs from the animal health anti-helmintic area have been examined in the above models, no new leads have been identified. It is assumed that due to the fact that the currently relevant drugs used for management of animal parasitosis are threatened by resistance, the "for profit" research based drug discovery companies must have several new compounds in the "pipeline". Unfortunately, due to the high potential commercial value these are not being made available for evaluation against the human parasites. Project management 19. The MACROFIL activities are conducted within the operational frame of Product Research and Development of TDR. The Drug Discovery Research group manages activities related to compound screening and discovery of new targets. Evaluation of progress and decisions regarding scientific validity are performed by a Steering Committee composed of experts in the area of drug discovery. The clinical activities and product development are conducted through Product Development Teams, which are assessed by TDR PRD Research & Development committee. 20. During the reporting period the project has operated with a budget of US $ $1.71m. This represents a reduction of $390 000 compared to previous years. This reduction is reflected in significant lessening of drug discovery activities. Since Filariases R & D products are entering into late phase development, it is expected that the budgetary requirements will increase substantially. With the anticipated termination of OCP at the end of 2002 and uncertainty of future funding, it is difficult to plan and prioritise long term R & D activities.I