Tetracyclines Chloramphenicol Aminoglycosides Macrolides

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1 Protein synthesis inhibitors Antimicrobial protein synthesis inhibitors Joseph K. Ritter, PhD Assoc. Prof Department of Pharmacology and Toxicology MSB Room Tetracyclines hloramphenicol Aminoglycosides Macrolides Three distinct stages of protein synthesis Structure of bacterial vs. mammalian ribosomes Initiation Elongation - most classes of antibacterials act at this step Termination - Bacteria 30S + 50S 70S Mammalian cytoplasmic 40S + 60S 80S Mammalian mitochrondrial 30S + 50 S 70S Formation of an initiation complex

2 Mechanism of protein synthesis: the elongation cycle Protein synthesis inhibitors A site 3 1 Binding of next amino acyl trna to A site Aminoacyl trna + GTP + EFs 2 Transfer of nascent peptide from to aminoacyl trna on A site Tetracyclines hloramphenicol Aminoglycosides Macrolides Translocation f nascent peptide From A site back to (GTP + EF) transpeptidation mediated by Peptidyl transferase activity of the ribosome Structural formulas of the tetracyclines: five currently available Tetracyclines bind to the 30S subunit block the first step of the elongation cycle 1 Binding of next amino acyl trna to A site Aminoacyl trna + GTP + EFs General antimicrobial spectrum of tetracyclines ( Broad, Bacteriostatic ) Gram positive aerobes Streptococcus spp. Staphylococcus spp. Gram negative aerobes Enterics (E. coli, Shigella, Salmonella, Klebsiella, Enterobacter, Serratia, Proteus, etc.) Haemophilus influenzae Neisseria (cocci) Anaerobes Atypical bacteria (lack cell walls) Rickettsiae (RMSF) Mycoplasmas (pneumonia) (an intracellular organism!!!!) hlamydiae (pneumonia, STDs) Basis for selectivity of tetracyclines Tetracyclines bind selectively to bacterial ribosomes Activity requires that bacteria are able to take up and concentrate tetracyclines inside the cell

3 Pharmacokinetics Absorption ral formulations Impaired by divalent metal cations bioavailability differences (more lipophilic Tcs such as M and D have higher F, effect on gut superinfections? ) Distribution Readily cross cell membranes into cells and across lipid barriers, Vds approach ~ total body water oncentrate in developing bones and teeth oncentration in skin, gingival space Elimination Routes of elimination and half-lives vary with the agent Minocycline and Doxycycline are metabolized by the liver and half the longest half lives, others are eliminated via urine (GF) Tetracyclines Not Absorbed Well With Milk products Kaopectate Pepto-Bismol Vitamins containing iron alcium supplements Safety precautions: tetracyclines Effects on bone, teeth Incorporated into calcifying structures Use during enamel calcification has two effects Enamel hypoplasia Permanent discoloration Yellowish fluorescent stain Darkens with age and exposure to light Also shown to reduce bone growth rate onsidered a teratogen Should not be used in pregnant or nursing mothers (Ts cross placenta and into breast milk) Primary teeth affected if exposed during last half of pregnancy Permanent teeth affected if used in children 2 mo. to 7-8 years Safety precautions: tetracyclines Effects on bone, teeth Gastrointestinal effects Direct irritation (diarrhea) Superinfection (diarrhea with blood/mucus) Related to broad spectrum, oral bioavail. F) Less serious (. albicans) More serious (. difficile, antibiotic associated colitis, secrete protein toxin) Vestibular toxicity (minocycline only) Hepatotoxicity-fatty liver more common in pregnant women and in liver disease Renal- Fanconi syndrome outdated tetracyclines ommercially available tetracycline formulations Tets -not considered first line drugs for common infections, activity is too broad, invites development of resistance Tets are drugs of choice for: Mycoplasma pneumonia Rickettsiae Borrelia sp (burgdorferi and recurrentis) Brucella sp Leptospira Vibrio cholerae Meningococcal carrier state (minocycline) Helicobacter pylori (tetracycline) Dental use: periodontal disease (strips, hollow fibers, bands)

4 Resistance to tetracyclines hloramphenicol (hloromycetin ) Three mechanisms Inactivating enzymes (plasmid encoded factors) Factors that result in increased extrusion from cells (plasmid encoded factors) Mutations in the gene for the 30S ribosomal subunit General cross-resistance occurs for agents in the T class 2 N H H 2 H H H N H Hl 2 hloramphenicol binds to the 50S subunit and blocks the transpeptidation step of the protein synthesis elongation cycle A site 3 Translocation f nascent peptide From A site back to (GTP + EF) 1 Binding of next amino acyl trna to A site Aminoacyl trna + GTP + EFs 2 Transfer of nascent peptide from to aminoacyl trna on A site transpeptidation mediated by Peptidyl transferase activity of the ribosome Antimicrobial spectrum of chloramphenicol ( Broad, bacteriostatic ) similar to Tc s Gram positive aerobes Streptococcus spp. Staphylococcus spp. Gram negative aerobes Enterics (E. coli, Shigella, Salmonella, Klebsiella, Enterobacter, Serratia, Proteus, etc.) Haemophilus influenzae Neisseria (cocci) Anaerobes Atypical bacteria (lack cell walls) Rickettsiae (RMSF) Mycoplasmas (pneumonia) (an intracellular organism!!!!) hlamydiae (pneumonia, STDs) Mammalian vs. bacterial ribosome selectivity of chloramphenicol Not as selective as tetracyclines Inhibitory effect on mitochrondrial protein synthesis inhibits synthesis of cytochrome c oxidase, ubiquinone cytochrome c reductase, and proton translocating ATPase Absorption hloramphenicol pharmacokinetics Very lipophilic agent 100% oral F Distribution Vd = 0.95 liters/kilogram body weight similar to tetracyclines with one difference (reaches therapeutic conc. in SF) Elimination Phase 2 drug metabolism (conjugation to glucuronide metabolite, decreased glucuronide forming capacity in young and old)

5 Adverse effects of chloramphenicol Hematologic toxicity (bone marrow) Anemia, leukopenia, thrombocytopenia (predictable, due to effect on mitochondria) Aplastic anemia (unpredictable, genetic factors) Gray baby syndrome Result of chloramphenicol accumulation in newborns Mechanism of bacterial resistance to chloramphenicol Result of plasmid-encoded factor AT AT = hloramphencolacetyltransferase 2 N H2 H H H H N H Hl 2 AT Acetyl oa 2 N H H 2H H H NH Hl 2 H3 Binding to 50S ribosomal subunit Therapeutic uses of chloramphenicol (BENEFIT vs RISK consideration) 1. Typhoid fever (Salmonella typhi) 2. Bacterial meningitis H. influenzae B. fragilis (anaerobe) 3. Bacterial meningitis* Pneumococcus/meningococcus 4. Rickettsial infection* *hloramphenicol is considered a second-line agent for these uses Aminoglycosides = Amino sugars linked to an aminocyclitol ring via glycoside bonds Gentamicin 1 H 3 H H 2 N H H 3 H H

6 H 3 H H 2 N H H H 3 H Aminoglycosides Structures of four aminoglycosides Gentamicin (Garamycin) Tobramycin (Nebcin) Amikacin (Amikin) Netilmicin (Netromycin) Neomycin (Mycifradin) Streptomycin Spectinomycin (Trobicin) Three effects of aminoglycosides on protein synthesis (result of binding to the 30S subunit) Inhibit initiation Premature termination Errors in translation Aminoglycosides have narrower antimicrobial spectrum Gram positive aerobes Gram negative aerobes (enteric rods) 1. Klebsiella 2. Proteus 3. E. coli 4. Enterobacter 5. Pseudomonas 6. Serratia No activity toward anaerobes No activity toward atypical bacteria No activity in infections involving intracellular organisms Basis for selectivity of aminoglycosides--bacteria versus mammalian cells Mammalian cells H 3 H H 2 N H H 3 H H High polarity, high molecular weight Bacterial cells uter membrane 2 dependent, Active process ytoplasmic membrane Pharmacokinetic characteristics of aminoglycosides Absorption Unable to cross membranes Zero oral bioavailability Must be administered parenterally (IV,IM) for systemic effects, oral formulations are considered topical Distribution Unable to cross membranes Low volume of distribution (Vd = 0.3 liters/kg body weight = plasma +interstitial fluid = extracellular body water) excluded from NS concentrations in most tissues are low except for renal cortex and lymphatic fluid in the ear Elimination Unable to cross membranes No metabolism Excretion in unchanged form by glomerularfiltration (T1/2 = 2,3 hrs in individuals with normal kidney function) ( hrs in anephric patients)

7 haracteristics of toxicity associated with aminoglycosides Low therapeutic index-requires therapeutic monitoring Nephrotoxicity (10-25% of patients) oncentration in proximal tubular cells Proportional to plasma concentrations, more frequent during long term treatment, and in individuals with compromised renal function totoxicity oncentrate in inner ear fluids Tinnitus leading to hearing loss, chronic vertigo hearing loss, chronic vertigo reversible in early stages Neuromuscular blockade, apnea (high doses only) Bacterial resistance to aminoglycosides Streptomycin-rapid development-due to mutation in ribosomal binding protein thers slow development Mechanisms Failure of drug to permeate microbe (strict anaerobes, facultative anaerobes) Inactivation by R factor-encoded enzymes transmitted by plasmids (Gram negative enterics) MAJR Amikacin and netilmicin are less susceptible Structure of the R1 plasmid Km IS1 r-determinants Ap Sm, Su Replication genes and origin R1 (94kbp) m IS1 Transfer functions resistance transfer factor m = hloramphencol acetyltransferase Therapeutic uses of aminoglycosides 1. Pseudomonas aeruginosa 2. Systemic infections due to gram negative enterics 3. Enterococcal endocarditis due to S. viridans or S. faecalis (combined with penicillins) 4. Tuberculosis (streptomycin) 5. Topical or oral use for gastrointestinal tract sterilization (neomycin) 6. N. gonorrhea (spectinomycin)