1. Chemotherapeutic Agents

2. Definitions Chemotherapeutic agent:any chemical used to treat disease Antimicrobial agent:any chemical used to treat infection Antibiotic:compound produced by one micro- organism that kills/inhibits another Antiseptic:antimicrobial that is too toxic for internal use Disinfectant:chemical so toxic that it is used only on inanimate objects

3. The Golden Rule “ The right drug for the right bug ”

4. Ideal Antimicrobial Agent Soluble in body Stable in body Selectively toxic Consistent Toxicity Non-allergic Bacterial resistance difficult to develop Long shelf life Reasonable cost

5. Antibiotic a chemical substance produced by certain molds and bacteria that kills or inhibits the growth of another microorganism –Bacteriostatic inhibits –Bacteriocidal kills Altough initially isolated as natural products of microorganims, can now also be synthesized in the lab

6. Antibiotic producers Fungi Bacillus species Actinomycetes

7. Discovery of Penicillin Flemming –in 1928 observed inhibition of Staphylococcus by Penicillium mold 1930’s –Clinical trials of penicillin 1940’s –Developed mass production of penicillin due to need for it in World War II S. aureus Penicillium

8. Bacillus sp. –Gram-positive –Spores-formers

9. Streptomyces member of the bacterial order Actinomycetales –most successful genus in Streptomyces –Over 500 species Actinomycetes –Gram-positive bacteria. –resemble fungi, but are true bacteria prokaryotic cells unlike eukaryotic fungal cells. –non-motile, filamentous –form spores from aerial filaments distinct from bacterial endospores. Produce numerous antibiotics Streptomyces species are found worldwide in soil

10. Terms/Concepts of Antimicrobial Agent Selective Toxicity Spectrum of Activity Mode of Action Side Effects Resistance

11. Selective Toxicity Concentration that eliminates pathogen –Therapeutic dosage level Concentration that causes damage to host –Toxic dosage level Chemotherapeutic index = Maximum tolerable dose (per Kg body weight) Minimum therapeutic dose (per Kg body weight) __ High for Penicillin (bacteria) –low for heavy metal compounds (worm parasite)

12. Spectrum of Activity Range of microorganisms that are affected by agent –Broad spectrum Wide range, e.g. both gram-pos & gram-neg Used when infective bacterial agent on is not precisely identified –Narrow spectrum Limited number, or specific group of bacteria Used to prevent development of resistance Less of an affect on normal bacterial flora

13. Antibiotic Spectrum Obligate intracellular microorganisms Chlamydia – tiny, non-motile, spherical bacteria Rickettsia – small, non-motile, gram-negative bacteria

14. Antibiotic Mode of Action 1. 1.Cell Wall 2. 2.Cell membrane 3. 3.Protein synthesis 4. 4.Nucleic Acid Synthesis 5. 5.Antimetabolites

15. Some clinically important antibiotics AntibioticProducer organismActivity Site or mode of action PenicillinPenicillium chrysogenumGram-positive bacteriaWall synthesis BacitracinBacillus subtilisGram-positive bacteriaWall synthesis Polymyxin BBacillus polymyxaGram-negative bacteriaCell membrane Amphotericin BStreptomyces nodosusFungiCell membrane ErythromycinStreptomyces erythreusGram-positive bacteriaProtein synthesis NeomycinStreptomyces fradiaeBroad spectrumProtein synthesis StreptomycinStreptomyces griseusGram-negative bacteriaProtein synthesis TetracyclineStreptomyces rimosusBroad spectrumProtein synthesis VancomycinStreptomyces orientalisGram-positive bacteriaProtein synthesis RifamycinStreptomyces mediterraneiTuberculosisProtein synthesis

16. Bacteria have a high internal osmotic pressure Without a sturdy cell wall, this pressure will cause membrane to burst Antibiotics can interfere with formation of the cell wall Results in cell death by cell bursting open Cell Wall Disruption

17. Penicillin has a 4-member ring “looks like” part of the cell wall to the cross-linking enzyme Penicillin competes with the normal cell wall component for the cross-linking enzyme, i.e. competitive inhibition Prevents this enzyme from cross-linking cell wall penicillin Cell Wall

18. Penicillin Penicillin G is the natural penicillin –Produced by Penicillium notatum Administered by injection –because is degraded by stomach acids Rapidly absorbed into blood & rapidly excreted Used against: streptococcus, meningococcus, pneumonococcus, spirochetes, clostridia, aerobic gram- positive rods, some staphylococcus and gonococcus Active in urine; so used for urinary tract infections Generally nontoxic Problems –Allergic reaction (~5% in adults) –Bacterial resistance

19. Semi-synthetic Penicillins Add a side-chain to the penicillin structure Alters: –Chemical characteristics –Spectrum of activity –Development of bacterial resistance Methicillin –Penicillinase resistant –resistance by an different mechanism developed Ampicillin –broad spectrum ( gram-neg & gram-pos) –acid resistant, i.e. oral administration

20. Cell Wall - Polypeptide Antibiotics Bacitracin –Produced by Bacillus licheniformis –Small polypeptide –Inhibits cell wall formation –Used on lesions & wounds because: Poorly absorbed in body Toxic to kidneys Vancomycin –Streptomyces –Very narrow spectrum –Used against Staphylococcus that is resistant to penicillin –Vancomycin resistance is now developing

21. Cell Wall - Antimycobacterial Isoniazid (INH) –Inhibits synthesis of mycolic acid in cell wall of Mycobacteria Tuberculosis –Administered with other antibiotics to prevent development of resistance Ethambutanol –Inhibits incorporation of mycolic acid into cell wall –Weak drug– just used secondarily to avoid development of resistance to INH Rifampin (inhibits mRNA synthesis) –Hits alternative target in cell

22. Isoniazid Complications –Competitive inhibitor of Vitamin B 6 –Prevents enzymes from converting Vitamin B 6 to useful molecules –Often supplement patient’s diet with extra Vitamin B 6 during treatment

23. Cell Wall - Antifungals Echinocandins –First new class of antifungals in 40 years Fungal cell walls contain glucans (polysaccharide) –Echinocandins inhibit synthesis of glucans –So inhibits synthesis of fungal cell wall

24. Cell Membrane Disruption Act as detergent Distort cell membrane –Bind to phospholipids Membrane loses function –Selective transport Especially effective against gram-negatives –because of their high lipid content in outer membrane Can adversely affect host cells

25. Polymyxins From soil bacterium, Bacillus polymyxa Targets prokaryotic membrane Usually for skin infections by gram-negatives Toxic so used internally only under close medical supervision –Kidney damage –Respiratory arrest

26. Gramicidin Bacillus brevis Gram-positive infections Topical ointment –highly toxic – –cannot be administered internally – –used only on the skin as a lotion or ointment. Interacts with bacterial membrane to form channel Interferes with membrane function increases the permeability of the bacterial cell membrane

27. Antifungal antibiotics Many used for topical application only, due to toxicity for superficial fungal infections only Systemic fungal infections –Can be toxic to the animal host –Toxicity due to adverse effect on specific tissues kidneys, bone marrow

28. Amphotericin B Streptomyces inhibits membrane function of fungi –selective transport –cell shape binds to sterols in the cell membranes of fungi –degradation of membrane integrity and cell lysis –Fungal cell membrane contain ergosterol –Animal cells contain cholesterol

29. Inhibit Nucleic Acid Synthesis DNA synthesis inhibition –Metronidazoles –add functional groups to DNA –Makes DNA weak and prone to strand breakage –antibacterial and antiprotozoal drug –Amebiasis, giardiasis, Helicobacterer pylorii (ulcers) –Side effects: nausea, vomiting, diarrhea –Nalidixic acid & quinolines –specifically bind to enzymes necessary for DNA synthesis –DNA gyrase, that uncoils and coils the DNA –Prevents DNA replication –Bacteria die out

30. Inhibit mRNA synthesis - Transcription Rifampin –Streptomyces –binds to bacterial RNA polymerase –blocks bacterial DNA from transferring its information to RNA –Used in U.S. for TB and meningococcus treatment –Can cause liver damage

31. RNA synthesis disruption - Antifungals Flucytosine –Analogue of cytosine –Fungi have enzyme to convert Flucytosine to 5-Flurouracil –5-Flurouracil is incorporated into RNA in place of cytosine –Disrupts protein synthesis because: Either incorrect amino acid or no amino acid is put into protein protein synthesis stops

32. Protein Synthesis - Translation RNA translation to protein –bacterial ribosomes read the mRNA code –bring in the tRNA which holds the anti-codons –Carries in proper amino acid to add to polypepetide Antimicrobial agents –target the large and small subunits of bacterial ribosomes. –because ribosomes of prokaryotes are different from those of eukaryotes (70S vs 80S) Therefore specific to prokaryotic ribosomes Less toxic to eukaryotic host

33. Peptide chain elongation Streptomycin, kanamycin, tetracycline & neomycin –bind to the smaller 30S ribosomal subunit, Chloramphenicol, erythromycin & streptogramins –bind to the 50S ribosome –Streptogramins used with vancomycin-resistant pathogens Can be toxic to kidney & inner ear (ringing) Tetracycline stains teeth Chloramphenicol can damage bone marrow –Aplastic anemia low RBC’s & platelets count

34. Anti-metabolites Sulfonilamides, or Sulfa drugs –First antimicrobial agent Folic acid analogue (= similar structure) –Coenzyme necessary for metabolic pathways –Similar structure to a precursor of folic acid Competitive inhibition –Inhibit synthesis of folic acid Humans require their folic acid in their diet –humans do not synthesize folic acid –So human metabolism is not inhibited by sulfonilamides Allergies can develop PABA Sulfanilamide

35. Antiviral Purine & pyrimidine analogs Inhibit viral replication The most effective ones are incorporated more rapidly into viral-infected cells than uninfected cells Purine vidarabine pyrimidine idoxuridine

36. Acyclovir a guanosine analog Acyclovir is phosphorylated by viral enzyme –becomes a “false nucleotide” –Viral enzyme has a higher affinity for acyclovir than does host enzyme If false nucleotide is used by DNA polymerase –will halt DNA synthesis

37. Acyclovir and Thymine Kinase Nucleoside kinase nucleoside nucleotide Nucleoside kinase AcyclovirAcyclovir phosphate DNA Polymerae DNA Synthesis stops

38. Interferon Naturally synthesized by infected host cell Prevents surrounding cell from being infected Helps to limit infection

39. Antiprotozoal Quinines –Malaria; Plasmodium vivax –Interfers with protein synthesis –Especially red blood cells –Prevents parasite from metabolizing hemoglobin as an energy and carbon source Metranidazole –causes breakage of DNA strands –Trichomonas infections, Giardiasis,

40. Antihelminthic Niclosamide –Interfers with carbohydrate metabolism –tapeworms Mebendazole –Interfers with glucose uptake –roundworms Piperazine –Neurotoxin Paralyzes muscles of worm Can cause convulsions –pinworms

41. Side Effects Toxicity Allergy Disrupt Normal Flora –Superinfection –Overgrowth –More likely with broad spectrum antibiotics Resistance

42. Antibiotic resistance New enzymes –Degrade or alter antibiotic Alter Target – can’t bind won’t work –Ribosomes, enzymes Alter membrane –Prevent transport in –Actively pump out Bypass affected pathway by using an alternative pathway

43. Destroy Antibiotic

44. Penicillin resistance Bacteria obtains gene for production of the enzyme lactamase Breaks covalent bond of the lactam ring of penicillin

45. Resistance by ribosome alteration A bacterium alters the antibiotic's target –the 50S ribosomal subunit. –The drug is no longer able to bind to the ribosome –Can’t block protein synthesis. Altered ribosome Normal ribosome

46. Alter antibiotic’s transport protein

47. Efflux Mechanism– Pump Antibiotic out

48. Development of Antibiotic Resistance

49. Transferring Antibiotic Resistance

50. Increase in Antibiotic Resistance

51. Antibiotic Resistance

52. Basis of microbial resistance Inherent (Natural) Resistance –Streptomycete has gene which is responsible for resistance to own antibiotic –Gram-negative outer membrane (permeability) –lack of transport system (Xanthomonas) –lack of target (structure or biochemical reaction)

53. Basis of Microbial Resistance Acquired resistance (previously sensitive) –change in bacterial genome mutation and selection (vertical evolution) –spontaneous exchange of genes between strains and species (horizontal evolution) –conjugation –transduction –transformation

54. Medical problem of Drug Resistance development of resistance is correlated with level of antibiotic use –overuse has increased the incidence of and selection for resistance-conferring mutations

55. Examples of overuse/misuse prophylactic use before surgery empiric use (no known etiologic agent) increased use of broad spectrum agents antibiotics in animal feeds pediatric use for viral infections patients who do not complete course (TB,AIDS)

56. Spread of Resistance day-care, nursing homes, correctional facilities fecal-oral (sanitation, animal feeds) sexual transmission respiratory transmission international travel immunosupression (cancer, organ transplants)

57. How to control the problem Use a different antibiotic –we are running out (vancomycin resistant S. aureus) Modify antibiotic to circumvent resistance –organisms keep “catching up” Use antibiotic combinations –“double edged sword”

58. Other strategies reduce consumption of antibiotics –physician education –animal feeds use more targeted antibiotics –broad spectrum, “magic bullet” is not the answer patient education - take the entire treatment course

59. Other Strategies New Drugs –currently about 100 antibiotics on market –three main mechanisms of action inhibition of protein synthesis inhibition of cell wall synthesis/maintenance inhibition of DNA replication

60. Other strategies New drugs –pharmaceutical industry putting resources back into discovery, SAR –discoveries in microbial physiology and genetics offering new targets –combinatorial chemistry - mass screening –liaisons with university researchers –starting to show success

61. New Antibiotics Cell Wall inhibitors - based on beta lactam skeleton –carbapenems - broad spectrum imipenem (Primaxin) usually given with cilastatin (prevents degradation by renal enzymes) meropenem (Zeneca) - enhaced anti-Pseudomonas activity –penems - beta lactamase stable Aztreonam (Bristol-Myers Squibb) Moxalactam (Lilly)

62. New Antibiotics New Quinolones - result of SAR –Sparfloxacin (Rhone/Poulenc) –Clinafloxacin (Parke-Davis) Glycylcyclines –derivatives of tetracyclines –resistant to efflux mechamisms

63. New Antibiotics Drugs to inhibit resistance mechanisms –better beta lactamase inhibitors –blockers of tetracycline pump –Isoniazid resistance blocker (M.tb) Drugs which interfere with membranes –knowledge of membrane structure and function increasing

64. Conclusions Resistance to antimicrobials is currently and will continue to be a problem Safe and effective new antimicrobials are needed now and will continue to be needed New understanding of microbial physiology and genetics is presenting many new targets Pharma resources flowing back into antibiotic development Competition is high

65. Antibiotics summary   Triad of chemotherapy   Selective toxicity   Mechanisms of action: post-antibiotic effect   Resistance: use “big guns” cautiously   Eliminate misuses, e.g., use of antibacterials for viral infections   Need to discover and develop new antibiotics; antibiotics in amphibian and human skin