Anti TB Drugs and identifying lead compounds for future drug devrlopment


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Anti TB Drugs and identifying lead compounds for future drug devrlopment

  1. 1. Evaluation of fractions of selected African plants for antimicrobial activity against Mycobacterium tuberculosis Richin John Koshy, Nadir Khalil, Marie Larson, Henrik Andersson, Johanna Raffetseder, Elsje Pienaar, Daniel Eklund, Asaad Khalid and Maria Lerm Masters in Medical Biosciences Degree Project (45 ECTS),
  2. 2. Historical Background  Neolithic Time  2400 BC - Egyptian mummies spinal columns  460 BC  Hippocrates, Greece  First clinical description: Phthisis / Consumption (I am wasting away)  500-1500 AD  Roman occupation of Europe it spread to Britain  1650-1900 AD  White plague of Europe, causing one in five deaths 2
  3. 3. Discovery of Mycobacteriumtuberculosis  24th March 1882 (Robert Koch) TB Day  Discovery of staining technique that identified Tuberculosis bacillus  Definite diagnosis made possible and thus treatment could begin  1890 (Robert Koch)  Tuberculin discovered  Diagnostic use when injected into skin 3
  4. 4. Mycobacteria  Small, rod-shaped, aerobic bacilli  Present in the environment, soil and water  High concentration of lipids and wax in the cell wall  M tuberculosis grows quite slowly and colonies do not appear on solid media up to 2.5 -5 4
  5. 5. TransmissionThe spread of M. tuberculosis involves a 3-step process: Transmission of bacteria, Establishment of infection, and Progression to disease. Netter atlas of Human Anatomy 5
  6. 6. Pharmacological discoveries • 1946 p-aminosalicylic acid • 1948-1950 – Combination of Strep. and PAS • 1952 Isoniazid. – Replaces sanatorium as major treatment – Patients can be treated as out- patients • 1954 Pyrazinamide. (PZA) 6
  7. 7.  1956-1960  Combination therapy of INH and PZA cures TB 1955 Cycloserine 1962 Ethambutol 1963 Rifampicin 1970-1977  Combination of Rifampicin and Isoniazid adopted as International regime for treatment of TB 7
  8. 8. World Health Organization (WHO) and theInternational Union Against Tuberculosis andLung DiseasesWHO and other agencies replaced the term primary resistance by the term“drug resistance among new cases” and acquired resistance by the term“drug resistance among previously treated cases 8
  9. 9.  Drug resistance in mycobacteria is defined as a decrease in sensitivity to a sufficient degree that the strain concerned is different from a sample of wild strains of human type Multi-drug resistant tuberculosis is resistance to Isoniazid and Rifampicin. It is incorrect to classify a patient has having multi-drug resistant disease if they have an infection with a bacterium susceptible to Rifampicin but resistant to many other drugs XDR TB is resistant to all first line drugs namely; Isoniazid and Rifampin and Three or more second line drugs (SLD’S) that are used to treat MDR- TB No drugs available to treat resistant strains since none has been developed in the last 40 years 9
  10. 10. Epidemiology of MDR TB • 490,000 new cases of MDR-TB each year, with >110,000 deaths1 • Accounts for 5% of 9 million new cases of TB2 • MDR-TB rates higher than ever (up to 22.3%), particularly in former Soviet Union countries • XDR-TB reported by as many as 49 countries • Recent WHO/IUATLD Global Surveillance report indicated 7.5% (301/4012) of MDR TB to be XDR4 • Around 40,000 XDR-TB cases emerge every year1 10
  11. 11. Mechanisims of Drug Resistance inTuberculosis Lipincott Textbook of Pharmacology 11
  12. 12. Reasons identified for resistance Deficient or deteriorating TB control programmes resulting in inadequate administration of effective treatment Poor case holding, administration of sub-standard drugs, inadequate or irregular drug supply and lack of supervision Massive bacillary load, illiteracy and low socio-economic status of the patients, the epidemic of HIV infection, laboratory delays in identification and susceptibility testing of M. tuberculosis isolates 12
  13. 13. Impact of resistanceHuge individual as well as public health consequences in terms of Increased mortality Prolonged periods of infectiousness with increased risk of transmission of resistant pathogens to others Indirect costs (prolonged absence from work, etc) Increased direct cost (longer hospital stay, use of more expensive 2nd or 3rd line drugs 13
  14. 14. Drug Discovery Traditionally used plants are a valuable source for the discovery of modern drugs Screening of Chinese herbal medicine led to introduction of artemisinin from the leaves of Artemisia annua which is used worldwide for treatment against malaria Shifting the screening strategy from a single enzyme targets to a whole bacterial cell level have been showing successful results as it has a holistic approach and only gives hits that can enter the bacterium through the cell wall 14
  15. 15. Preliminary studies  Rosmarinus officinalis L (RO)  (Flowers/Leaves)  Antimicrobial  Antiviral 15
  16. 16. Preliminary studies  Khaya senegalenesis leaves (KSL)  Khaya senegalenesis barks (KSB)  Fever remedy  Antimicrobial  Antiprotozoal 16
  17. 17. Hypothesis Fractions of Khaya Senegenalenesis and Rosimarinus Officinalis L possess anti-microbial propertiesAIM To identify fractions of plant extracts used traditionally to treat infectious diseases in African countries for possible antimicrobial activity against M tuberculosis 17
  18. 18. Materials and methods Mycobacterial culture Infection of human Fractions of Plant macrophages with Extracts prepared M tuberculosis with DMSO Cytotoxicity with Analysis of hMDM and bacterial growth THP-1 cells by lumometry THP-1 cell culture Determination of CFU plating Separation and culture of primary monocytes 18
  19. 19. 19
  20. 20. Monocytes were cultured in Monocytes arecomplete DMEM medium and diffrentiated intoincubated at 37ºC. macrophages. 20
  21. 21. Bacteria cultured and OD Monocytes were cultured in Macrophages infected withmeasured complete DMEM medium and bacteria at MOI 10 and incubated at 37ºC. Monocytes fractions of extracts diffrentiated into macrophages 21
  22. 22. Results: Assesing Mycobacterial growth using Luminometry in the presence of RO fractions (0.00625 mg/ml) 22
  23. 23. Assesing Mycobacterial growth using Luminometry in thepresence of Khaya senegalenesis bark and leaves[KSB/KSL] (0.00625 mg/ml) 23
  24. 24. Viability of Macrophages treated with RO and KSB fractions(10, 1 and 0.1 mg/ml) 10 mg/ml 1 mg/ml 0.1 mg/ml 24
  25. 25. Viability of THP-1 macrophages treated with RO and KSBfractions (10, 1 and 0.1 mg/ml) 10 mg/ml 1 mg/ml 0.1 mg/ml 25
  26. 26. Viability of infected macrophages treated with RO and KSBfractions (0.1 mg/ml) 26
  27. 27. Assessing intracellular Mycobacterial growth using Luminometryin the presence of Rosimarinus officinalis [RO] (0.1mg/ml) 27
  28. 28. Assessing intracellular Mycobacterial growth using Luminometryin the presence of selected Khaya senegalenesis bark [KSB](0.1mg/ml) 28
  29. 29. Conclusion Plant extract fractions of K Senegalenesis and R Officinalis were analysed for anti-mycobacterial activities which yielded four fractions to have distinct anti-mycobacterial properties. High-throughput screening using luciferase determines mycobacterial growth giving conclusive results. Inhibition of growth by these extracts was observed in both systems giving inference about their antituberculous activity which appears to be meaningful. Biologic actions of K Senegalenesis have been attributed to a complicated concert of compounds for its synergestic and antagonistic activity. This has enabled the pharmacological and biological properties of the plant in exerting its anti-microbial properties. Further phytochemical studies need carried out in order to find the active compound with inhibition. 29
  30. 30. Acknowledgement First I will like to give my greatest appreciation to my supervisor Maria Lerm for her direction, encouragement and support throughout the period of my project. I am happy that I had the opportunity to work under her supervision and ever thankful for all that she taught me A big thanks to Nadir for everything and keeping good company I also express my gratitude to Johanna Raffetseder, Marie Larsson, Elsje Pienaar and Daniel Eklund for introducing me to most of the methods i used in this project and more and also for always being there to answer numerous questions I had. Finally but not the least, I will like to say a big thanks to all the staff of the department 30
  31. 31. Thank You