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08/01/2018

WEEK WEEK WEEK 27 27 27 Overview – Slide Set 3 The Bacterial Cell ( Prokaryotic )

SULPHONAMIDES OSPAP Programme • Agents targeting the cytoplasm TRIMETHOPRIM – [Sulfonamides] NITROFURANTOIN – Trimethoprim – Nitrofurantoin • Agents targeting the ribosomes MPHM14 Antibacterial – – Chemotherapy – – – • Agents targeting the nuclear material – Rifamycins – Fluoroquinolones • Updates and new developments • Clinical applications

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WEEK WEEK WEEK 27 27 27 Agents acting in the cytoplasm Sulfonamide Antibacterials Folic Acid Synthesis

• Many common biochemical processes in cytoplasm of all cells • Sulfonamides were the first synthetic antibacterials used clinically • Rely on differences between prokaryotic and eukaryotic cells for • Bacteriostatic action selectivity • – Agents targeting metabolic processes only found in bacteria provide Sulfonamides are structural mimetics of para -aminobenzoic acid selective antibacterials (PABA, essential metabolite) and compete for a key enzyme in the PABA • Bacteria synthesise several vitamins, e.g. folic acid, not synthesized synthesis of folic acid in mammalian cells • Folic acid is an essential metabolite for mammals (cannot be – These processes are good targets for antibacterial agents synthesised by mammalian cells), so this interference in folic acid • The sulfonamides are antifolates (like methotrexate) and interfere synthesis is the basis of the selectivity of the sulfonamides with the bacterial biosynthesis of folic acid • Sulfonamides are extremely rarely used now due to extensive • Trimethoprim is also an antifolate, targeting a different part of the resistance in both G+ve and G-ve bacteria Folic acid Dihydropteroic acid folic acid biosynthetic pathway – Alterations to the enzyme target, dihydropteroate synthase (DHPS) Sulfonamides resemble PABA and • Nitrofurantoin acts on several processes in the cytoplasm, some – Mutations to gene for DHPS resulting in poor sulfonamide binding similar to mammalian processes, but… are mistakenly used instead, • Only sulfamethoxazole (as Co-trimoxazole) and sulfadiazine in BNF preventing synthesis of folic acid – Is prodrug and only activated efficiently in bacterial cells

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WEEK WEEK WEEK 27 27 27 NH2 Co-trimoxazole (Septrin) UTIs OMe Trimethoprim N • Although sulfonamides rarely used now, co-trimoxazole is sometimes H2N N OMe still useful for urinary tract infections OMe – Susceptibility should be established 1 st [culture / MS methods] • Trimethoprim is an antifolate agent through dihydrofolate reductase • Also useful for: (DHFR) inhibition; bacteriostatic – Pneumonia due to Pneumocystis jirovecii (fungus, was called Pneumocystis carinii ) – • Used for UTIs (good renal as unchanged drug), acute and Although reduction of DHF to THF also occurs Toxoplasmosis and nocardiasis in mammalian cells, trimethoprim has about • chronic bronchitis (well distributed), pneumocystis pneumonia 40,000x greater affinity for the bacterial Co-trimoxazole associated with rare, serious ADRs, especially in elderly enzyme than the mammalian version – hence patients • Can be used as monotherapy or in combination (synergistic) with the selectivity – Was replaced by amoxicillin or cephalexin, but less commonly used now due to sulfamethoxazole (Co-trimoxazole) in ratio 5 : 1 resistance (amoxicillin) and increased risk of C. diff infection after use (cephalexin) – Similar gives best combination • Nitrofurantoin more commonly used now • Co-trimoxazole acts on two enzymes in the same biosynthetic – (Relatively) broad spectrum, bactericidal agent, particularly against E. coli , most sequence (sequential blocking ) Enterococci , Klebsiella sp., Staphylococci and Streptococci • Doses of both drugs are lower than would be required if either used (But poor activity vs Proteus sp. and inactive vs Pseudomonas sp.) alone so side-effects (and possibly resistance) can be minimised – Rapid renal elimination after oral admin: low serum, high urinary concentration

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WEEK WEEK WEEK 27 27 The Bacterial Cell ( Prokaryotic ) 27 Agents targeting protein synthesis via ribosome Nitrofurantoin MoA

• Activated by several bacterial enzymes to reactive nitrosyl radical • Reacts with cytoplasmic proteins and other macromolecules • Inhibits: protein synthesis, aerobic () energy metabolism, DNA synthesis, RNA synthesis, and cell wall synthesis • Resistance rare, even although introduced into clinic in 1952; resistance may be due to non-specific activity – resistance would require concurrent changes to many enzymes, proteins and pathways = unlikely • Although broad spectrum, rapid and efficient absorption from GI tract reduces risk to GI bacteria (low risk of C. diff resulting)

Nitrosyl radical CHLORAMPHENICOL TETRACYCLINES MACROLIDES AMINOGLYCOSIDES LINEZOLID http://whyfiles.org/126dna_forensic/images/dna.gif

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WEEK WEEK WEEK 27 27 27 Transcription and translation Transcription and translation - revision Chloramphenicol (Chloromycetin, Cm) TRANSCRIPTION Transcription and • Genetic code on DNA template transcribed into messenger RNA by RNA polymerase • Originally obtained from venezuelae , translation describe the • In bacteria, mRNA moves to ribosomes [ cf in eukaryotes, mRNA exits nucleus into cytoplasm] now prepared synthetically • processes by which the TRANSLATION Bacteriostatic with broad spectrum of activity; only R,R -isomer is active genetic code on DNA is • Ribosomes have two subunits: 30S and 50S in bacteria [70S] (40S and 60S in eukaryotic cells [80S]) • Highly lipophilic and penetrates most tissues converted into proteins • mRNA binds to small subunit of ribosome, followed by initiator: tRNA-methionine (crosses blood-brain barrier) In bacteria, these Replication • Large ribosome subunit binds to small subunit. Large ribosome subunit has two binding sites, P • Active against Neisseria meningitidis , Streptococcus processes all take place in Translation and A in the centre (PTC) pneumoniae and Haemophilus influenzae (causes of meningitis) the cytoplasm, making an • DNA Transfer RNA (tRNA) – amino acid Transfer RNAs carry amino acids to the ribosome site where mRNA binds (charged tRNA) • Can be used in treatment of meningitis in patients with β-lactam allergies and easy target for • Only tRNA with codon of complementary sequence to that on mRNA can bind to ribosome drug of choice against typhoid fever antibacterial agents Transcription Codon = 3 nucleotides (triplet), which codes for a specific amino acid • But…. severe toxicity possible, so only given systemically in life-threatening Selectivity is possible, as • Ribosome moves along mRNA from 5′ to 3’: once peptide bond formed, non-acylated tRNA leaves infections: benefit must outweigh risks Messenger RNA (mRNA) RIBOSOME (rRNA) bacterial ribosomes are P site and peptide-tRNA moves from the A to the P site • Side-effects after systemic admin include aplastic anaemia (bone marrow cannot different to mammalian A new charged tRNA-amino acid (as specified by the mRNA codon) enters A site replenish blood cells) – unpredictable and may occur weeks after treatment ribosomes • Peptide chain grows as amino acids added until stop codon reached, then leaves ribosome through ceases Protein http://highered.mcgraw- protein exit tunnel • Commonly used topically in treatment of bacterial conjunctivitis hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120077/micro06.swf::Protein%20Synthesis

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WEEK WEEK WEEK 27 27 27 Chloramphenicol: MoA & resistance Resistance to chloramphenicol Linezolid (Zyvox ®)

• Resistance to chloramphenicol in Pseudomonas aeruginosa is due to efflux pumps (via both chromosomal and plasmid transmission) • First member of oxazolidinone agents • Chloramphenicol binds to large ribosome subunit (50S) at the peptidyl • An RNA methyltransferase (cfr ) methylates a key part of the 23S subunit – Only example currently licensed transferase centre A site, preventing binding of the next charged tRNA near to ‘fenicol’ binding site and blocks the binding • New synthetic class of antibacterials • www.youtube.com/watch?v=0VINqUF-r5I • Resistance also arises due to chloramphenicol acetyltransferases (CAT) • • Discovered in 1990s, approved in 2000 Binding involves 4 H-bonds from chloramphenicol to the ribosome and which acetylate chloramphenicol so that it no longer binds to the PTC A 2+ • Active against G+ve bacteria, such as: coordination to a Mg ion in the catalytic site site. CAT genes are both plasmid (e.g. CatC in S. aureus ) and – MRSA • Selectivity arises due to differences between the conformations of chromosome-derived (e.g. CatB3 in Salmonella typhimurium ) – Community acquired and nosocomial pneumonia (caused by G+ve bacterial and eukaryotic PTC • does not contain 3-OH so is not acetylated. Cm-resistant strains bacteria) • Chloramphenicol inhibits bacterial protein synthesis as it only binds to A in which resistance is due to CATs are susceptible to florfenicol site of bacterial ribosome – Vancomycin-resistant Enterococcus faecium (VRE) • However, use of systemic chloramphenicol can lead to serious side effects • Not active against G-ve bacteria due to inhibition of mammalian mitochondrial protein synthesis • Bacteriostatic to Enterococci and Staphylococci; bactericidal to Streptococci Inactive Acetylated chloramphenicol • Time dependent, low PAE

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WEEK WEEK WEEK 27 27 27 Mode of Action Resistance to Linezolid Tetracyclines

• Also inhibits protein synthesis, but at different point to other agents • Resistance 1st noted in 1999 (before approved!) • Binds to pocket formed by 8 RNA base residues, conserved across all • Still relatively rare, probably due to restricted use of linezolid Generic Name (Old) Trade Name X R1 R2 R3 bacterial 50S ribosomes • In 2013, >98% Staph aureus sensitive to linezolid • Causes slight change in conformation to rRNA, so tRNA units have reduced (Achromycin / Sustamycin) H OH Me H affinity to A site • Acquired resistance by 2 mechanisms: (Declomycin) Cl OH H H

• Also inhibits formation of 70S unit by blocking interactions between 50S and – Mutation in 23S rRNA unit at linezolid binding site Oxymycin / (Terramycin) H OH Me OH 30S subunits – Methylation of 23S rRNA by methyltransferase (also causes resistance to (Aureomycin); not licensed Cl OH Me H • Selectivity for bacterial ribosomes, little inhibition of mammalian chloramphenicol and ) Doxlar / Vibramycin H H Me OH cytoplasmic protein synthesis • Both change the linezolid binding site and prevent its binding Aknemin NMe HHH • But side effects due to inhibition of mitochondrial protein synthesis linked to 2 • G-ve bacteria have intrinsic resistance due to endogenous efflux pumps myelosuppression if extended period of use Lymecycline* Tetralysal H OH Me H – (prodrug of tetracycline) – Cf toxicity of systemic chloramphenicol – similar issue Prevents sufficient linezolid getting into cells to have antibacterial effect *Amide NH 2 (at RHS) is modified to NHCH 2NH(CH 2)4CH(NH 2)CO 2H

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WEEK WEEK pKa 9.4 WEEK 27 27 27 General Information Pharmacokinetics pKa 3.3 • Broad spectrum agents against G-ve and G+ve bacteria and other organisms,

e.g. chlamydiae, mycoplasmas, rickettsia Tetracycline • In general, tetracyclines considered bacteriostatic pKa 12.0 pKa 7.5 N-tButylglycine Minocycline – But certain tetracyclines bactericidal against specific bacteria if used at • • appropriate concentration The oral of tetracyclines varies according to the species Developed recently to combat tetracycline resistance (FDA approved 2005) • First glycyltetracycline (based on minocycline, which is rarely used itself due – Doxycycline with quinine can be used to treat/prevent malaria – Doxycycline and minocycline have high oral bioavailability (>90%) to ADRs); others in development (e.g. , in phase III CT) • – Lower oral bioavailability for other members (50 – 75%) Time dependent, but also active in concentration-dependent manner (PK-PD • Indicated for skin, soft tissue and intra-abdominal infections caused by MDR type III) – Lymecycline is more aqueous soluble: 5000x more than tetracycline, and bacteria – Concn dependent parameters give good clinical results (T at concn above MIC) is absorbed by active transport from GI tract, yet still only ca. 50% • Administered IV (why?) available • • Strong PAE; t½ 7 – 17 hours Active against many G+ve and G-ve bacteria, including: – Absorption of most is affected by food (absorption decreased) – • Well distributed throughout tissues and body fluids; variable protein binding MRSA, VRE, Haemophilus influenzae, Neisseria gonorrhea , MDR – Any complexed to metal ions in GI tract are excreted in faeces Acinetobacter baumanii and tetracycline-resistant bacteria • For most tetracyclines, dosing 1-2 daily is sufficient – Non-absorbed tetracyclines can disrupt commensal bacteria and result in – Active at about 5 µg/mL • Less commonly used now due to increased levels of resistance gastric disturbance – Inactive against Pseudomonas and Proteus species

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WEEK WEEK WEEK 27 27 Dehydration under acid conditions: 27 Tetracyclines Tetracyclines

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microbewiki.kenyon.edu/images/thumb/1/17 /Congenital.jpg/300px-Congenital.jpg • Bacteriostatic • Chlortetracycline discovered (in 1948) from Streptomyces aureofaciens Unstable to acid • Drugs of choice for Chlamydia trachomatis infections (e.g. urethritis). • Tetracycline obtained either by hydrogenolysis of chlortetracycline or Used to treat , and respiratory and genital infections, e.g. syphilis and fermentation of S. alboniger in chlorine-free medium chlamydia, and Lyme disease • Derive their name from tetracyclic ring system (octahydronaphthacene) • Bind to the ribosome 30S subunit through H-bonds and Mg 2+ chelation, • More stable Dehydration in acid conditions at C5a-C6 leads to inactive to acid inhibits binding of the aminoacyl-tRNA and leads to termination of anhydrotetracyclines (demeclocycline more stable than chlortetracycline) peptide chain growth • Second generation tetracyclines, doxycycline and minocycline, and third • Poor affinity for eukaryotic ribosome = basis of selectivity generation, tigecycline, more stable to acid as no C6-OH to be protonated • Resistance causing decreased use of these agents and arises due to: (C6 = CHMe or CH ) 2 – tetracycline efflux systems, e.g. Neisseria gonorrhoeae (chromosomal • 3+, 2+ 2+ Much more Chelate polyvalent metal ions (Fe Ca and Mg ) so should never be given stable to acid transmission) and S.aureus (plasmid transmission) with dairy products, or co-administered with iron-rich antacids, and not – ribosomal protection proteins, e.g. S. aureus (plasmid transmission) given to children under twelve – enzymatic deactivation of tetracyclines, e.g. by TetX (anaerobic bacteria)

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WEEK WEEK WEEK 27 27 Resistance by Enzymatic Deactivation of 27 Macrolides (e.g. ) Tetracycline binding to 30S rRNA Tetracyclines • indicates large lactone (ester) Binding through H-bonds (to G1053, C1054, ring ; these also contain 2 G1198, U1196, C1195, A965, G966) , sugar units – desosamine and cladinose aromatic stacking (with C1054), and • Erythromycin [ R=H ] (14-membered ring) magnesium ion chelation (phosphates of C1054, first isolated from Saccharopolyspora G1197 and G1198 with Tet Os on C11 & C12) erythraea in 1952 by Eli Lilly • Erythromycin is bacteriostatic with broad antibacterial spectrum, similar to that of penicillins so is alternative for penicillin- allergic patients • Eythromycin is mildly basic ( pKa 8) due to aminosugar group and usually administered as the HCl salt Others in the family: • • Prodrug esters, e.g. Erythromycin ethyl [ R1 = Me] • succinate [ R=CO(CH ) CO Et ] 2 2 2 • See later (Erythroped), are used to mask taste of bitter drug

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WEEK WEEK WEEK 27 27 27 Acid instability of Erythromycin Azithromycin • Lactone ring extended by 1C Macrolides: PK-PD • Acid instability of erythromycin overcome by use of enteric-coatings so • No ketone at C9 , so greater acid that drug is only released in the higher pH (7-8) of the small intestine stability • Bacteriostatic activity with low post- effect • • ca 40% oral bioavailability and good • Water insoluble dosage forms (e.g. stearate salt [CH 3(CH 2)16 CO 2H] also tissue distribution Similar spectrum of activity to β-lactams, often substituted for penicillin if allergic used to overcome this problem and bitter taste (insoluble salts: do not – • t 70-90h Active vs most G+ve bacteria (e.g. Staph and Strep ) and some G-ve dissolve in saliva, so no taste) ½ – • Less G+ve activity, but increased vs G- Also active against mycobacteria ve, incl. H. influenzae • Erythromycin and clarithromycin: time dependent, low PAE (Type II) – 2-4 times daily • Azithromycin: time (& concn) dependent with good PAE (Type III) – once daily dosing Telithromycin • Telithromycin: concentration dependent, PAE 0.5 - 4hrs (Type I) – once daily dosing • New form – cladinose at C2 • Macrolides have better oral bioavailability than expected, perhaps due to unknown replaced by ketone and side arm at C11 active transport across GI tract? • Substituted OMe at C6 and O • Excellent tissue distribution means can be used for skin and soft tissue infections, but at C12, so greater acid stability poor ability to cross BBB • Oral bioavailability 57% • Macrolides accumulate in phagocytes (e.g. macrophages): transported to site of • t½ 10h bacterial infection and released • Similar activity spectrum – Concentrates amount of macrolide at site of infection • The other macrolides, azithromycin, clarithromycin and telithromycin, are to erythromycin, but – Effect particularly strong with azithromycin much more acid stable, e.g. azithromycin: 10% degraded after 20 minutes active vs penicillin and erythromycin • Clarithromycin rapidly metabolised, but to active metabolite (R 1 = OH) at pH2 (and 37 oC) vs 4 seconds for erythromycin resistant S. pneumoniae Slide 31 of 75 OSPAPMPHM14 Antineoplastic Chemotherapy Slide 32 of 75 OSPAPMPHM14 Antineoplastic Chemotherapy Slide 33 of 75 OSPAPMPHM14 Antineoplastic Chemotherapy

WEEK WEEK WEEK 27 27 27 Macrolides: mode of action Resistance to Macrolides Aminoglycosides • Resistance arises due to modifications to the ribosome , increased • Macrolides block exit of protein tunnel by binding • Aminoglycosides currently in UK clinical use: to a high affinity site, leading to the arrest of expression of efflux pumps , and chemical inactivation of the – protein elongation and the dissociation of macrolide – shortened peptidyl-tRNAs from the ribosome 1. Inducible or constitutive erm (erythromycin ribosome methylase) gene – • Main component of binding pocket is nucleotide gives rise to resistance in Streptococci – 2058. In bacteria, this is adenine (A) and In Streptococcus pneumoniae , ribosomal methylase dimethylates a single (semi-synthetic version) macrolides bind strongly to this nucleotide. In site, A2058 (on N-6), resulting in a decreased binding affinity for • (1 st in series) discovered in 1944 erythromycin due to the increased size of this nucleotide eukaryotic cells, this nucleotide is guanine (G) and is • Isolated from an actinomycete mould, Streptomyces griseus • i.e. change to target too bulky to allow favourable interactions with the • First agent to cure pulmonary tuberculosis 14-membered macrolides  selectivity 2. In Campylobacter jejuni (cause of food poisoning), over-expression of macrolide efflux system causes resistance • Many other aminoglycosides from Streptomyces family • Desosamine sugar (formation of 3 hydrogen bonds – Neomycin from S. fradiae in 1949 between C2′-OH and A2058 and A2509, NMe and 3. Two different types of chemical inactivation can occur: 2 • – A2505), ring hydroxyls (hydrogen bonds between Phosphorylation or glycosylation of C2’-OH desosamine ring prevents H- Kanamycin from S. kanamyceticus in 1957 (M. Gaynor and A.S. Mankin, bonding to rRNA the 6, 11 and 12-OH and nucleotides), and lactone Frontiers in Med. Chem ., 2005, 2, 21) – Tobramycin from S. tenebrarius in 1968 • Enterobacteriaceae express esterase that hydrolyses lactone ring to give – Gentamicin, was isolated in 1963 from various species of (hydrophobic interactions) play key roles in inactive acyclic product macrolide binding to this site bacteria, as a complex of several structurally-related agents

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WEEK WEEK WEEK 27 A note on names….. 27 27 Mode of Action • Rapid bactericidal action, concn dependent: almost independent of bacterial • The end of the name of an , -mycin or –micin, relates to the load if above MIC microorganism source of each agent: • Broad spectrum vs G-ve and G+ve – Aminoglycosides from Streptomyces species given the ending –mycin • Very polar molecules – small amount enters cells – from a Micromonospora species have the ending –micin • Initially bind (H-bonds) to 16S ds rRNA part of 30S subunit adjacent to A site • Useful guide to the origin of an aminoglycoside, but…. • Causes slight conformation change to A site, resulting in mis-reading of tRNA – Not necessarily extended to naming of other microorganism-derived agents and incorrect amino acids added to growing protein chain: produces – Many examples of agents from Streptomyces species not named by same system, ‘nonsense’ proteins e.g. chloramphenicol, novobiocin, cycloserine, and clavulanic acid. • Alters function of bacterial membrane with loss of membrane semi- permeability, difficult to repair • Structurally, antibiotic aminoglycosides are glycosidic polycyclic structures, • incorporating an aminocyclitol ring: Allows influx of large concentrations of aminoglycoside • Halts protein synthesis entirely: fatal to bacterial cells – Streptamine, streptidine or 2-deoxystreptamine, • Selective for bacterial cell protein synthesis, but not entirely even at low • 2-Deoxystreptamine most commonly found in clinical aminoglycosides concns: at v high concns, eukaryotic protein synthesis also halted • Two or three aminosugar rings are usually linked to the aminocyclitol • Widespread use limited by ototoxicity and nephrotoxicity

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WEEK WEEK WEEK 27 27 27 Resistance Amikacin The Bacterial Cell ( Prokaryotic ) • SULPHONAMIDES  1. Most common cause of resistance to aminoglycosides is due to R Amikacin is a successful example TRIMETHOPRIM of synthetic modification to factor-mediated enzymes β-LACTAMS VANCOMYCIN  – TEICOPLANIN These modify the aminoglycoside structure: • Designed to reduce susceptibility ISONIAZID • N-acetylation to metabolism and avoid resistance • O-phosphorylation • • L-Hydroxyaminobutyryl amide O-adenylation chain inhibits deactivation by – Structural modifications prevent binding to rRNA adenylation and phosphorylation, even at remote positions 2. Resistance can also arise due to point mutations in rRNA A site • Enhanced potency and spectrum – Due to specific single nucleotide residue changes – Active against Pseudomonas – Aminoglycoside no longer binds effectively to rRNA POLYMYXINS aeruginosa resistant to other QUINOLONES DAPTOMYCIN  CHLORAMPHENICOL agents RIFAMYCINS 3. Resistance also due to decreased aminoglycoside uptake into TETRACYCLINES  – Active against Mycobacterium MACROLIDES bacterial cells AMINOGLYCOSIDES tuberculosis LINEZOLID – Due to decreased cell membrane permeability and/or increased efflux http://whyfiles.org/126dna_forensic/images/dna.gif

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WEEK WEEK WEEK 27 27 27 (Fluoro)Quinolone Antibacterials Example (F-)quinolone antibacterials Pharmacokinetic considerations • All FQs have good oral bioavailability (despite their zwitterionic nature), • Nalidixic acid was discovered in 1962 during the synthesis and probably due to lipophilic groups. Lower oral bioavailability for earlier purification of chloroquine (anti-malarial). generation FQs • Nalidixic acid and other first generation quinolones have weak anti- – Ciprofloxacin (1 st generation) bioavailability: 70% bacterial (bactericidal) activity – Levofloxacin ( 3 rd generation) bioavailability: 99% • • Early quinolones active against G-ve bacteria only, due to poor uptake LogP app (at pH 7.0) -1.6 (ciprofloxacin)  +2.5 (nalidixic acid), by passive diffusion into G+ve bacteria (use porins for G-ve uptake) Levofloxacin logP app 2.1 • Later generations more potent and broader spectrum, including vs • Concn-dependent antibacterials, require high serum concentrations for G+ve bacteria, mostly due to the introduction of a fluorine at the 6- effective activity; PAE 2-6 hours

position and substituent at C5 (see slide 9) = ‘FQs’ • Dosing depends largely on t ½: longer half-life (& PAE) allows once daily admin • nd rd th th st Now have 2 , 3 and 4 (5 ?) generation fluoroquinolones – t½ 3-5 hours for 1 generation fluoroquinolones

• Bactericidal effect – t½ longer for later generation fluoroquinolones, e.g. 7.4 hours for levofloxacin and • Concentration dependent with prolonged PAE 9.6 hours for moxifloxacin • 1st generation used in higher doses due to protein binding and weaker activity

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WEEK WEEK WEEK 27 27 27 Applications Side effects Structural requirements for activity • FQs active primarily against G-ve bacteria + some G+ve bacteria – e.g Ciprofloxacin: broad spec vs G-ve (+ many G+ve), partic Salmonella, Shigella, • Common side-effects include GI disturbance, rashes, fatigue, Campylobacter, Neisseria ; used for RTI, UTI, GIT + bone/joint infections. Not v dizziness, visual disturbances active vs Strep. pneumoniae + Enterococcus faecalis (G+ve) • If used alongside NSAIDs, convulsions can occur • st nd 1 (+ some 2 ) generation excreted mostly unchanged in urine, have been • Other serious side effects: used to treat UTIs (eg nalidixic acid, norfloxacin) – Spontaneous tendon ruptures – Used less commonly now, due to increased resistance – Prolongation of the QT interval (eg moxifloxacin) • 3rd and 4th generation FQs reported to be significantly more lipophilic, • Probably through blocking of the hERG channel better absorbed (90 -100%), and widely distributed after oral, ophthalmic or (Human cardiac potassium channel = human ether a-go-go channel) respiratory system delivery – These side effects can be chronic and severe; some fatalities • Greater versatility for treating systemic infections: • Long QT syndrome led to several FQs being withdrawn from market, – Superior activity across a wider range of Gram positive bacteria eg – Improved volume of distribution and uptake into many cells • Hundreds of analogues synthesized and evaluated – Grepafloxacin (4 th generation) in 1999 • – Enhanced lipophilicity enables, e.g. treatment of prostatic and respiratory FQ structure activity relationship well established – rd infections and infections in the brain / CSF Sparfloxacin (3 generation) in 2001 • Fewer new analogues being made now (but still some research to improve efficiency, reduce side effects and try to evade resistance mechanisms) – NB Most staphylococci resistant to FQs: not active vs MRSA Slide 46 of 75 OSPAPMPHM14 Antineoplastic Chemotherapy Slide 47 of 75 OSPAPMPHM14 Antineoplastic Chemotherapy Slide 48 of 75 OSPAPMPHM14 Antineoplastic Chemotherapy

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WEEK WEEK WEEK 27 27 27 Metallic interactions Fluoroquinolone Targets Resistance to fluoroquinolones (FQs)

• Bactericidal • Resistance to the FQs arises through two major mechanisms: • Structure of FQ essential for binding to gyrase (the target), but • Targets: inhibition of bacterial DNA gyrase and topoisomerase IV 1. Alterations in the target enzymes : DNA gyrase and /or topoisomerase IV also allows binding of other species • The right handed helical nature of DNA means that positive supercoils – Alterations to the DNA gyrase occur via mutations in the quinolone-resistance • Rich O donor ligand area : quinolone carbonyl and carboxylic (knots) form ahead of replication sites when DNA strands act as templates determining region (QRDR) of the gyrA gene which encodes the two A subunits of the tetrameric enzyme (gyrB encodes the two B subunits) acid for new strands • – Similar mutations have been described in topoisomerase IV • Co-admin with metal-ion containing preparations In order for DNA replication to proceed, these supercoils must be removed by the gyrase or topoisomerase IV relaxing the DNA chain. By • These alterations to the target enzymes result in decreased FQ leads to significant reduction in oral absorption catalysing the formation of negative supercoils, these enzymes remove binding • Chelates range of metal ions: the positive supercoils and give a tension free DNA double helix 2. Decreased accumulation of the FQs in cells due to the impermeability of the membrane ( decreased uptake ) or the over-expression of efflux pumps – Mg 2+, Al 3+ , Zn 2+ , Ca 2+, or Fe 3+ • DNA gyrase and topoisomerase IV relax bacterial DNA by cutting one of – FQs cross the outer membrane via specific porins (G-ve; all quinolones) or – No co-admin with metal-cation the strands, passing the other strand through the cut and then resealing the cut diffusion through the phospholipid bilayer (G+ve; hydrophobic, newer containing agents, e.g. generation FQs only) • Mammalian cells do not have DNA gyrase or topoisomerase IV (they do antacid preparations, – Porins are protein channels which allow passive diffusion of a specific agent iron supplements, milk have topoisomerases I and II, but FQs do not bind to these enzymes); across the cell membrane hence, these agents show selectivity for bacterial enzymes

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WEEK WEEK WEEK 27 27 27 Specific examples Rifamycins Therapy / combination therapy • The rifamycins are antibiotics from Amycolatopsis mediterranei and include Rifampicin, Rifabutin and Rifapentine • Outer membrane of P. aeruginosa has very low permeability to • Rifampicin is used to treat: • Discovered in 1957; >100 semi-synthetic analogues made small hydrophobic molecules giving this bacterium intrinsic – tuberculosis and leprosy (both • Rifampicin is an effective semi-synthetic bactericidal agent resistance to the quinolones mycobacterium infections) • Others also in clinical use, e.g. rifabutin (long t : once daily dosing) • Rifater; with isoniazid and pyrazinamide • Its low permeability in part due to secretion of mucoid alginate ½ • Prolonged PAE: >65 hours in vitro • Rifinah; with isoniazid biofilm – MRSA / staphylococcal endocarditis (in – Gives rise to name: ‘blue-green pus bacteria’ combination with another antibacterial, • e.g. (active vs pen-resistant E. coli has three main porins and a decrease in the level of one of Enterococci) these (OmpF) is associated with an increase in resistance to the • Obtained from Fusidium coccineum : inhibits quinolones Added chemically protein synthesis • Ps. aeruginosa (Gram negative) and S. aureus (Gram positive) – Legionella pneumophila (Legionnaires’ disease) exhibit well characterised efflux pumps for the quinolones – Can be used as prophylactic therapy against meningococcal meningitis ( Neisseria meningitidis ) and Haemophilus influenzae

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WEEK WEEK WEEK 27 27 DNA-dependent RNA polymerase: 27 Rifampicin MoA Rifampicin resistance / interaction RNAP • Inhibitor of bacterial DNA-dependent RNA polymerase • Mutations to target : Mycobacterium tuberculosis resistance to • Prokaryotic RNA polymerase (RNAP) is a holoenzyme composed of rifampicin (rifampin) due to mutations in rpoB gene which the core enzyme and a σ factor (gives the polymerase specificity for codes for the β subunit of RNAP, resulting in a decreased a particular promoter region of DNA) affinity for rifampicin • • These mutations have no effect on growth rate of M. Rifampicin binds to a pocket in the β subunit of the complex α2ββ ′ω structure tuberculosis (naturally slow growing and can tolerate less • Amino acid sequence of rifampicin binding site is conserved among active transcription) bacterial RNAPs but not between bacterial and eukaryotic RNAPs • Interaction : inducer of cytochrome P450 enzymes so can lead (selectivity ) to increased metabolism of many drugs cleared through liver • Rifampicin blocks transcription once the RNA becomes 3 — this interaction leads to reduced efficacy of the oral nucleotides long through both allosteric and steric effects contraceptives and restricts choice of anti-HIV drugs

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WEEK WEEK WEEK 27 27 New agents: Ceftaroline fosamil 27 New antibacterial agents Ceftaroline fosamil SAR • There is a huge global need for new antibacterial agents, yet few Pos charged pyridinium provides pharmaceutical companies are interested zwitterionic counter charge • New cephalosporin derivative = prodrug (active parent: ceftaroline) – In Dec 2015, only 39 antibacterials in clinical trials in USA 1,2,4-thiadiazole ring enhances Oxime provides • • Potent in vitro activity due to very high binding affinity for PBP uptake into G-ve bacteria and There are many hurdles to developing new antibacterials, e.g. β-lactamase stability – Costs >$2.5 bn to develop 1 prescription-only med to market • Active against wide range of pathogenic bacteria: increased affinity for PBP – Approx. 20% success rate in phase I CT – high failure rate – G+ve : MRSA, MSSA, Streptococcus pneumoniae (incl. pen resistant), – Acute use: only 5-7 days dosing, not economic Streptococcus pyogenes (incl. macrolide resistant), Enterococcus faecalis – High risk of resistance developing: loss of market – G-ve : Haemophilus influenzae, Enterobacter cloacae, Proteus mirabilis, • Recent FDA and EMA incentives to increase pharma interest Shigella species • Most antibacterials in CT are new derivatives of existing classes (e.g. – cephalosporins, tetracyclines/aminocyclines, FQs) Not active vs VRE – • T : 2-3h; PAE 1.5 -7h depending on bacterial species 1,3-diazole ring Useful as increased activity, fewer side effects and lower resistance ½ Phosphoramidate prodrug confers MRSA activity • Not all bad news: some new classes of antibiotics • Of particular use for CAP and SSTI due to excellent tissue distribution enhances aq solubility – E.g. FabI inhibitors, , Teixobactin • Little development of resistance so far Hydrolysed in vivo to free amine Cephem ring system – inhibits PBP • However, little progress towards countering ESKAPE threat – Need new agents active against G-ve bacteria

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WEEK WEEK WEEK 27 27 27 New agent: MoA / Resistance? Current status • New semi-synthetic member of class of antibiotics – Originally isolated in 1950 from mould Pleurotus mutilis • Acts on ribosome to block protein synthesis • Strong in vitro data allowed progress to clinical trials – General structure solved in 1962 • Unique MoA : binds to specific site on 23S RNA of 50S bacterial ribosome • • Medicinal chemistry changes resulted in agents for veterinary use in subunit Phase II for cellulitis completed 2010: comparative to 1979 () and 1999 () – Inhibits ribosomal peptidyl transferase activity, and partially inhibits the vancomycin (but no resistance) binding of the initiator tRNA substrate to the ribosomal P-site – Unable to balance oral bioavailability and side effects with efficacy for humans • Now in phase III clinical trials in US and EU for CAP • Novel mechanism compared to other agents acting on ribosome means no • – developed as topical agent for humans (2007) cross-resistance observed with macrolides, tetracyclines, aminoglycosides Started Sept 2015, ends Aug 2017 • Recently, developed new derivatives and linezolid – Comparing efficacy and safety with moxifloxacin ( ± linezolid) with suitable PK for oral – Also not seen with fluoroquinolones, trimethoprim-sulfamethoxazole, • Phase III CT for skin and soft tissue infections (SSTI) planned administration in humans mupirocin and β-lactam agents – Successful phase II CT completed in 2011 – • Lefamulin (BC-3781) best candidate Lefamulin possesses potent in vitro activity against the most common • Not yet available in UK unless patients part of trial – Others in development pathogens associated with CAP: S. pneumoniae , H. influenzae , S. aureus , M. pneumoniae , L. pneumophila , and C. pneumoniae , including MDR strains • Altho old class, has in vivo activity against both G+ve and G-ve Derivatised part: semi-synthetic bacteria with novel MoA RS = HO in parent General pleuromutilin structure

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WEEK WEEK WEEK 27 27 27 New class of antimicrobials ☺ FabI inhibitors Choosing an antibacterial agent

• Many considerations to make, e.g. • Teixobactin: Ling et al ., Nature, 2015 • Fab = fatty acid biosynthesis, an essential pathway for growth in – • both G+ve and G-ve bacteria and Mycobacteria How severe is the infection? Agent required to kill bacteria (bactericidal) or 99% of bacteria are not able to be cultured in vitro but offer novel simply halt growth (bacteriostatic)? antibiotics • Fab inhibitors target the fatty acid synthase II (Fas-II) pathway that consists of several enzymes catalysing essential steps in fatty acid – Which bacteria are causing the infection? Which agent is active against that • New β-proteobacteria species, Eleftheria terrae , cultured in situ using biosynthesis pathogen? What is the susceptibility profile to that agent? new technique • Key regulatory reactions are most targeted by researchers: – Where is the site of the infection? Which agent has the correct distribution • Novel depsipeptide isolated with time-dependent bactericidal activity elongation condensing enzymes (FabF and FabB) and enoyl-acyl characteristics? • Very potent vs G+ve bacteria (at <1 μg/mL), including MRSA and VRE, carrier protein reductase (FabI) – What dosing method and regimen are required for effective treatment? C. diff, Bacillus anthracis and Mycobacterium tuberculosis • Several existing antibacterials found to act on Fas-II pathway, e.g. • Most effective treatment when use specific agent (not just class of • isoniazid (FabI), cerulenin (FabF), thiolactomycin (FabB and FabF), Teixobactin inhibits cell wall synthesis by binding to lipid II (precursor triclosan (FabI) agent) appropriate to: of peptidoglycan) and lipid III (precursor of cell wall teichoic acid) – • New agents being developed, 3 currently in CT in USA Pathogenic bacteria – use narrow spectrum if possible (susceptibility?) – • Attempts to create resistance so far failed – All targeted at Staphylococcus aureus and MRSA (e.g. Debio 1452) Site of infection – distribution of agent must be suitable to reach site • Good news, but….. not active against P. aeruginosa or K. pneumoniae ! – Little success currently in creating broad spectrum FabI inhibitors – Pharmacokinetic requirements – dosage method / regimen – And not orally bioavailable (peptide) 

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WEEK WEEK WEEK 27 Broad or narrow spectrum? 27 27 CAP Examples • Inflammation / swelling in the tissues of one or both lungs, usually • Some antibacterial agents are active against both Gram positive and result of bacterial infection Gram negative bacteria • Annual UK presentation rate in community: 0.5%-1% – β-Lactams (broad spectrum G-ve and many G+ve [except aztreonam]) • – 5-12% of adults who present to GPs with symptoms of lower RTI are Community acquired pneumonia diagnosed with CAP – Tetracyclines (broad spec G-ve and some G+ve) • – 22-42% of these are admitted to hospital (about 100,000 hospital – Chloramphenicol (broad spec vs both G+ve / G-ve; but severe side effects possible if Meningitis admissions each year in England) systemic admin) • C. diff infection • Mortality rate 5-14% [patient.co.uk (1 st Jan 2016)] – Macrolides (e.g. erythromycin: broad spec vs both G-ve / G+ve) • Common infective agents: – Aminoglycosides (G-ve and some G+ve) • TB – G+ve : Strept. pneumoniae (20-60% of bacterial CAP in adults; 15-40% in – Trimethoprim/sulfamethoxazole (G-ve and some G+ve) children) Staph. aureus (2% CAP; uncommon cause in healthy adults) – • Rifamycins (broad spec vs both G-ve and G+ve) Infective endocarditis – G–ve : Haemophilus influenzae (3-10% of CAP) • Other agents only active against one or other • UTIs • Agents causing atypical pneumonia: – Linezolid (G+ve only) – Mycoplasma pneumoniae , Chlamydophila pneumoniae (G-ve) and – Daptomycin (G+ve only) Legionella pneumophila (G-ve) [Cause up to 15% of CAP cases] • Treatment requirements: – Colistin (G-ve only; except cocci and certain other G-ve bacteria) – Broad spec activity vs G-ve and G+ve – Vancomycin (G+ve only; but severe side effects possible) – Good tissue distribution (so sufficient concentration reaches lungs)

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WEEK WEEK WEEK 27 27 27 CAP treatment HAP / VAP Meningitis-causing bacteria • Initial treatment: amoxicillin or doxycycline or clarithromycin • Can be bacterial or viral: bacterial usually most dangerous (about 50% • Amoxicillin: β-lactam antibiotic, bactericidal with good PAE, active vs • Early onset (2-4 days after admission): same as for CAP mortality if untreated) G+ve (Strep and Staph) and some G-ve (H. influenzae , but not Ps. • Assume community source prior to admission • Most common causes: aeruginosa ), good distribution into different tissues, time dependent • Late onset (≥5 days after admission): (T > MIC important), orally bioavailable – Gram +ve : Streptococcus pneumoniae; Listeria monocytogenes • • Doxycycline: tetracycline class, broad spectrum (G-ve and G+ve), G+ve : MRSA (10-15% nosocomial pneumonia – common 5d after – Gram –ve : Neisseria meningitidis ; Haemophilus influenzae type B (latter primarily bacteriostatic, time / concn dep, good PAE, well distributed start of viral influenza) in infants and children) throughout tissues, excellent oral bioavailability • G-ve : Ps. aeruginosa (PA; major cause of nosocomial pneumonia); • Severe and life threatening, rapid empirical Tx required – With loading regimen, follows concn dep kinetics, i.e. large dose = better Klebsiella pneumoniae (associated with recent use of potent • Usually 1st line: ceftriaxone effect antibiotics); Neisseria meningitidis (usually associated with – Without loading regimen, follows time dep kinetics and takes several – Broad spectrum G+ve and G-ve bacteria days to be effective meningitis and septicaemia, but can also cause pneumonia) – Admin IV for rapid action st • Clarithromycin: macrolide, bacteriostatic, low PAE, time dep, good oral • 1 line often pipericillin with tazobactam (Tazosyn) – High protein binding and low renal excretion give half-life of several hours bioavailability and tissue distribution, concentrated at site of infection, – Active against Ps. aeruginosa – Paracellular uptake (due to leaky meninges) and specific transport into CSF espec. lung, by accumulation in macrophages – If severe, aminoglycoside added, or Ceftazidime or FQ (e.g. ciprofloxacin) – Low active transport out of CSF leads to accumulation during meningitis • New agents also active: ceftaroline fosamil and lefamulin • If MRSA suspected: vancomycin (unless resistant) – Good PAE with G+ve bacteria and H. influenzae

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WEEK WEEK WEEK 27 27 27 Bacterial meningitis Summary Resources • Significantly affected by distribution of drug • TARGET Toolkit: – Distribution of drug across BBB into CSF important • Bacteria and bacterial infections – Must guarantee effective dose in CSF – http://www.rcgp.org.uk/targetantibiotics/ • Covered many classes of antibacterials • – Local guidelines: ‘basket of ten’ In meningitis, BBB compromised, so get higher than usual drug • concentrations in CSF Range of targets • Start Smart Then Focus: • Selectivity – • β-lactams (ionised), but reach about 5 – 20% of levels found in serum www.gov.uk/government/uploads/system/uploads/attachment_data/f ile/215308/dh_131181.pdf – 3rd generation cephalosporins (e.g. Ceftriaxone, Cefotaxime) have good BBB • Mode of action – Being reviewed and updated in 2015 penetration and active against most bacteria causing meningitis (unless resistant) • Mechanism(s) of resistance • Antimicrobial prescribing and stewardship competencies: • Lipophilic drugs reach higher concentrations • Bactericidal vs bacteriostatic – www.gov.uk/government/uploads/system/uploads/attachment_data/f – Fluoroquinolones, rifampicin, chloramphenicol: • Time dependent vs concentration dependent actions 30 – 50% of serum concentrations (even under normal circumstances), but FQs ile/253094/ARHAIprescrcompetencies__2_.pdf rarely used due to resistance • Guidelines, especially for emergency care – Being developed for implementation 2016 • For successful treatment of meningitis, require • New treatments, new agents, new classes • Mnemonics: http://www.cram.com/flashcards/mnemonics- – Rapid action • Resources galore-958558 – Concentration in CSF of 10 – 30 times MBC GOOD LUCK!

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