Linezolid Case Study

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  • testing scheme may seem exceedingly simple, but that is a reflection of the lack of significant knowledge of the biological activity of oxazolidinone when started the program
  • It should also be noted that these early analogs were prepared in racemic form in order toexpedite their preparation. Since only the 5-(S)-enantiomer is antibacterially active, the racemic material generallyexhibits half the potency of the pure enantiomer. Nevertheless, these racemic analogs were deemed sufficient toprobe SAR at this early stage of the program. By 1989 fragmentary reports were obtained that DuPont had abandoned their oxazolidinone program
  • The soft tissue infection data with linezolid provided two useful pieces of information. Linezolid was clearly penetrating tissues (in this case between the dermis and muscle layers) sufficiently in order to cure replicating pathogens and it was doing so when dosed orally. The oralroute of administration with this antibiotic was extremely impressive as the drug given orally was equivalent to subcutaneously-adminsteredvancomycin in bacteremia models and oral administration resulted in sufficient tissue levels to treat difficult soft tissue infections.
  • CFU=colony-forming units of bacteria
  • The rapidity with which linezolid kills the bacterial cells becomes irrelevant to the fact that it inhibits toxin production and thereby inhibits the tissue damage and destruction which is the hallmark of the disease.
  • it was determined that linezolid binds the 50S ribosomal subunit and that binding then prevents formation of a functional initiation complex
  • it was determined that linezolid binds the 50S ribosomal subunit and that binding then prevents formation of a functional initiation complex
  • it was determined that linezolid binds the 50S ribosomal subunit and that binding then prevents formation of a functional initiation complex
  • it was determined that linezolid binds the 50S ribosomal subunit and that binding then prevents formation of a functional initiation complex
  • it was determined that linezolid binds the 50S ribosomal subunit and that binding then prevents formation of a functional initiation complex
  • The morpholino ring of linezolid does not appear to make significant interactions with the ribosome, which is consistent with the fact that many different functional groups can be substituted for the morpholine without a significant loss of activity.
  • Linezolid binds the 50S A-site, near the catalytic center, which suggests that inhibition involves competition with incoming A-site substrates.(CCA-Phe), an analogue of the portion of aminoacyl and peptidyltRNAs, which bind nearly equally well to the H50S A- and P-sites.the binding conformation of linezolid in the presence of CCA-Phe does not differ from the conformation observed in the structure of linezolid alone
  • ribosomal elongation factor
  • it was determined that linezolid binds the 50S ribosomal subunit and that binding then prevents formation of a functional initiation complex
  • The rapidity with which linezolid kills the bacterial cells becomes irrelevant to the fact that it inhibits toxin production and thereby inhibits the tissue damage and destruction which is the hallmark of the disease.
  • The rapidity with which linezolid kills the bacterial cells becomes irrelevant to the fact that it inhibits toxin production and thereby inhibits the tissue damage and destruction which is the hallmark of the disease.
  • Linezolid Case Study

    1. 1. Linezolid – a true small molecule antibiotic<br />Alexei Pushechnikov, Ph.D.<br />Disney group<br />January 13, 2009<br />
    2. 2. Outline<br />Introduction<br />Synthetic Approaches<br />Activity<br />Mode of Action<br />Conclusions<br />2<br />
    3. 3. Introduction<br />3<br />E.I. du Pont de Nemours & Co.<br />Slee, A.M. et al 1987 <br />DuPont’s SAR of the oxazolidinonepharmacophore:<br />electron-withdrawing groups in the aryl para- position provided optimal activity <br />importance of the N-aryl group<br />additional substitutions at the aryl ortho- position or C-4 of the oxazolidinone ring had a detrimental or indifferent effect on the antibacterial activity<br />C-5 (S)-configuration required for antibacterial activity<br />optimal activity of a C-5 acetamidomethyl group<br />Ford, C. W.; Zurenko, G. E.; Barbachyn, M. R. Cur. Drug Targets2001, 1, 181-199<br />Barbachyn, M. R.; Ford, C. W. Angew. Chem. Int. Ed. 2003, 42, 2010-2023<br />Brickner, S.J.; Barbachyn, M.R.; Hutchinson, D.K.; Manninen, P.R. J. Med. Chem. 2008,51, 1981-1990<br />
    4. 4. Introduction<br />4<br />Barbachyn, M.R.; Brickner, S.J.; Hutchinson, D.K. WO95/07271 The Upjohn Company (Filed on April 1994) <br />Brickner, S.J.; Hutchinson, D.K.; Barbachyn, M.R.; Manninen, P.R.; Ulanowicz, D.A.; Garmon, S.A.; Grega, K.C.; Hendges, S.K.; Toops, D.S.; Ford, C.W.; Zurenko, G.E. J. Med. Chem. 1996,39, 673<br />
    5. 5. Introduction<br />5<br />Generalized testing scheme for oxazolidinones development<br />Analogues<br />In Vitro testing<br /> - Intrinsic activity<br /> - Activity against resistant strains<br /> - Spectrum of activity<br />SAR<br />In Vivo testing<br /> - Acceptable in vivo activity <br /> - Route of administration <br />Pharmacokinetics/Toxicology<br /> - Useful blood levels<br /> - Frequency of dosing <br /> - Acceptable toxicity profile <br />Further Biological Characterization <br /> - Mechanism of action<br /> - Pharmacodynamics<br /> - Tissue penetration <br />Clinical Trials<br />Registration<br />Barbachyn, M. R.; Ford, C. W. Angew. Chem. Int. Ed. 2003, 42, 2010-2023<br />
    6. 6. Introduction<br />6<br />Drug-like compounds (Lipinski’s rule)<br /> * Not more than 5 hydrogen bond donors : 1 <br /> * Not more than 10 hydrogen bond acceptors : 6 (8)<br /> * A molecular weight under 500 daltons : 337<br /> * A partition coefficient logP less than 5 : 0.9<br /> * Number of atoms from 20 to 70 : 44 <br /> * Number of rotatable bonds less than 10 : 5<br /> * Polar surface area (PSA) less than 140 : 91<br />Computed Properties - Chem3D Properties Broker<br />Linezolid<br />(PNU-100766)<br />Good solubility : 3.7 mg/mL in pH 7 phosphate buffer<br />The oral bioavailability : 100% (rapid and complete absorption)<br />The excretion : 20–30% of the dose found in the urine as the parent drug <br />Has been approved by the FDA in 2000 under the trade name Zyvox<br />Ford, C. W.; Zurenko, G. E.; Barbachyn, M. R. Cur. Drug Targets2001, 1, 181-199<br />Barbachyn, M. R.; Ford, C. W. Angew. Chem. Int. Ed. 2003, 42, 2010-2023<br />Brickner, S.J.; Barbachyn, M.R.; Hutchinson, D.K.; Manninen, P.R. J. Med. Chem. 2008,51, 1981-1990<br />
    7. 7. Introduction<br />7<br />
    8. 8. Introduction<br />8<br />Pharmacia&Upjohn’s revised SAR :<br />electron-donating nitrogen atom well tolerated and often improves safety profile <br />N-aryl group required for activity<br />fluorination of phenyl ring often improves antibacterial activity/efficacy <br />C-5 (S)-configuration necessary for antibacterial activity<br />C-5 acetamidomethyl group essential for good activity<br />
    9. 9. Synthetic approaches<br />9<br />Ford, C. W.; Zurenko, G. E.; Barbachyn, M. R. Cur. Drug Targets2001, 1, 181-199<br />Barbachyn, M. R.; Ford, C. W. Angew. Chem. Int. Ed. 2003, 42, 2010-2023<br />Brickner, S.J.; Barbachyn, M.R.; Hutchinson, D.K.; Manninen, P.R. J. Med. Chem. 2008,51, 1981-1990<br />
    10. 10. Synthetic approaches<br />10<br />Ford, C. W.; Zurenko, G. E.; Barbachyn, M. R. Cur. Drug Targets2001, 1, 181-199<br />Barbachyn, M. R.; Ford, C. W. Angew. Chem. Int. Ed. 2003, 42, 2010-2023<br />Brickner, S.J.; Barbachyn, M.R.; Hutchinson, D.K.; Manninen, P.R. J. Med. Chem. 2008,51, 1981-1990<br />
    11. 11. Synthetic approaches<br />11<br />Process scale synthesis<br />Ford, C. W.; Zurenko, G. E.; Barbachyn, M. R. Cur. Drug Targets2001, 1, 181-199<br />Barbachyn, M. R.; Ford, C. W. Angew. Chem. Int. Ed. 2003, 42, 2010-2023<br />Brickner, S.J.; Barbachyn, M.R.; Hutchinson, D.K.; Manninen, P.R. J. Med. Chem. 2008,51, 1981-1990<br />
    12. 12. Synthetic approaches<br />12<br />Lohray, B. B.; Baskaran, S.; Rao, B. S.; Reddy, B. Y.; Rao, I. N. Tetrahedron Lett. 1999, 40, 4855-4856<br />
    13. 13. Activity<br />13<br />In vitro activities of linezolid and vancomycin, Minimum Inhibitory Concentration (mg/L)<br />Barbachyn, M. R.; Ford, C. W. Angew. Chem. Int. Ed. 2003, 42, 2010-2023<br />
    14. 14. Activity<br />14<br />In vivo activities of linezolid and vancomycin, Effective Dose (mg/kg) <br />Linezolid – orally <br />Vancomycin - subcutaneously<br />Barbachyn, M. R.; Ford, C. W. Angew. Chem. Int. Ed. 2003, 42, 2010-2023<br />
    15. 15. Activity<br />15<br />Activities against Mycobacterium tuberculosis<br />In Vitro<br />In Vivo<br />Treatment was started 1 day after the mice received 7x106 viable mycobacteria.<br />Cynamon,M.H.; Klemens,S.P.; Sharpe,C.A.; Chase,S. Antimicrob. Agents Chemother. 1999, 43,1189-1191<br />
    16. 16. Activity<br />16<br /><ul><li>Linezolid lacked significant effects against most Gram-negative pathogens including E. coli, Klebsiellapneumoniae, and Proteus penni
    17. 17. Despite the observed in vitro activity, linezolid was ineffective against the Moraxellacatarrhalisand H. influenzae, even at concentrations above the MIC
    18. 18. However, in the absence of cell membranes and cell walls the oxazolidinones were very active in inhibiting E. coli protein synthesis
    19. 19. Making the E. coli transmembrane pump nonfunctional made whole E. coli cells sensitive to linezolid both in vitro and in vivo
    20. 20. The lack of Gram-negative activity is the result of the presence of transmembrane pumps which, along other molecules, pump oxazolidinones out of the cell faster than they can accumulate</li></li></ul><li>Activity<br />17<br />Pharmacokinetic Profile<br />
    21. 21. Activity<br />18<br />Linezolid behaves as a cidal drug in vivo although it is clearly static for staphylococci and enterococci in the test tube<br />Linezolid is generally considered to be well tolerated in humans. Most common side effects in the clinical trials were (percent incidence) : <br />diarrhea (2.8-11%) <br />nausea (3.4-9.6%) <br />headache (0.5-11.3%)<br />With longer term usage of linezolid (&gt;2 weeks), there is an association of reversible myelosuppression (anemia, thrombocytopenia, leukopenia, or pancytopenia)<br />Linezolid is a weak, reversible, and nonselective inhibitor of Monoamine Oxidase. A risk of serotonin toxicity is anticipated with linezolid. Avoidance of large quantities of food with a high tyramine level (aged cheese, beer, or red wine) along with administering linezolid is suggested<br />Lawrence, K.R.; Adra, M.; Gillman, P.K. Clin. Infect. Dis. 2006, 42, 1578-1583<br />Brickner, S.J.; Barbachyn, M.R.; Hutchinson, D.K.; Manninen, P.R. J. Med. Chem. 2008,51, 1981-1990<br />
    22. 22. Mode of Action<br />19<br />Swaney,S.M.; Aoki,H.; Ganoza,M.C.; Shinabarger,D.L. Antimicrob. Agents Chemother. 1998, 42, 3251-3255<br />Clemett,D.; Markham,A. Drugs 2000, 59, 815-827<br />
    23. 23. Mode of Action<br />20<br />[14C]Eperezolid binding to E. coli ribosomes. <br />(A) Total ribosomes; (B) 50S subunits; (C) 30S subunits. <br />▓, total binding; █, specific binding.<br />Lin,A.H.; Murray,R.W.; Vidmar,T.J.; Marotti,K.R. Antimicrob. Agents Chemother. 1997, 41, 2127-2131<br />
    24. 24. Mode of Action<br />21<br />Binding to ribosomes. <br />Eperezolid and linezolid bind to the 50S ribosomal subunit with Kd ~20 M<br />Competition by various concentrations of unlabelled antibiotics. <br />(A) [14C]eperezolid binding; (B) [14C]chloramphenicol binding. <br />●, eperezolid; ■, linezolid; ▲, chloramphenicol; ▼, lincomycin.<br />Lin,A.H.; Murray,R.W.; Vidmar,T.J.; Marotti,K.R. Antimicrob. Agents Chemother. 1997, 41, 2127-2131<br />
    25. 25. Mode of Action<br />22<br /><ul><li>Oxazolidinones block translation initiation, binding to a site on the 50S subunit closely related to the chloramphenicol and lincomycin binding site and near the interface with the 30S subunit.
    26. 26. The resulting distorted site may prevent the correct positioning of the 30S initiation complex from forming the 70S initiation complex and hence inhibit translation initiation.</li></ul>Lincomycin<br />Chloramphenicol<br />Lin,A.H.; Murray,R.W.; Vidmar,T.J.; Marotti,K.R. Antimicrob. Agents Chemother. 1997, 41, 2127-2131<br />Thompson, J.; O’Connor, M.; Mills, J.A.; Dahlberg, A.E. J. Mol. Biol. 2002, 322, 273–279<br />
    27. 27. Mode of Action<br />23<br /><ul><li> Location of linezolid resistance mutations in E. coli 23S rRNA(central loop of domain V and the neighboring regions).
    28. 28. E. coli linezolid resistance mutations are shown by arrows (thickness proportional to the level of linezolid resistance for each mutation).
    29. 29. Marked positions of nucleotide substitutions that confer linezolid resistance in H. halobium(boxed) and in S. aureusand E. faecalis(circled).</li></ul>Kloss,P.; Xiong,L.; Shinabarger,D.L.; Mankin, A.S. J. Mol. Biol. 1999, 294, 93-101<br />Xiong,L.; Kloss,P.; Douthwaite, S.; Andersen, N.M.; Swaney,S.; Shinabarger,D.L.; Mankin, A.S. J. Bacteriol.<br />2000, 182, 5325-5331<br />
    30. 30. Mode of Action<br />24<br />A<br />B<br />Orientation of Linezolid at the Peptidyltransferase Center of the Ribosome. <br />Model for the binding position of linezolid (Lnz, red) with respect to nucleotides (blue) at the E. coli PTC. <br />Relative position of linezolid (red) compared to chloramphenicol (Cam, green). PTC nucleotides are shown as green surface representation.<br />Leach, K.L., Swaney, S.M., Colca, J.R., McDonald, W.G., Blinn, J.R., Thomasco, L.M., Gadwood, R.C., Shinabarger, D., Xiong, L., Mankin, A.S. Mol. Cell2007, 26, 393-402<br />Wilson, D.N.; Nierhaus, K.H. Mol. Cell2007, 26, 460-462<br />
    31. 31. Mode of Action<br />25<br />Superposition of the structure of linezolid (cyan) with the structures of A-site (orange) and P-site (green) substrate analogues bound to H50S <br />Linezolid (cyan) and CCA-Phe (gold) binding to H50S. Linezolid molecule occupied the A-site and CCA-Phe occupied the P-site <br />(PDB code 3CPW)<br />Ippolito, J.A.; Kanyo, Z.F.; Wang, D.; Franceschi, F.J.; Moore, P.B.; Steitz, T.A.; Duffy, E.M. J. Med. Chem. 2008, 51, 3353-3356<br />
    32. 32. Mode of Action<br />26<br />The binding site of oxazolidinones.<br />Linezolid bound to the Deinococcusradiodurans50S ribosomal subunit.<br />Oxazolidinones induce an A/O state recognized by LepA. Relative position of linezolid (red), P-tRNA (cyan), A-tRNA (pale green), LepA (maroon density and ribbon) and A/L-tRNA (blue). <br />Wilson, D.N.; Schluenzen, F.; Harms, J.M.; Starosta, A.L.; Connell, S.R.; Fucini, P. Proc. Natl. Acad. Sci. USA 2008,105, 4673-4678<br />
    33. 33. Mode of Action<br />27<br />Events during normal translation (A–D), compared with the effect of the linezolid (red) during translation (E–H).<br />Wilson, D.N.; Schluenzen, F.; Harms, J.M.; Starosta, A.L.; Connell, S.R.; Fucini, P. Proc. Natl. Acad. Sci. USA 2008,105, 4673-4678<br />
    34. 34. Conclusions<br />28<br />Linezolid has been approved in the U.S. <br />for the treatment of nosocomial and communityacquired pneumonia caused by S. aureus(methicillin-susceptible or MRSA) or S. pneumoniae(penicillin-susceptible or multidrug-resistant strains) and vancomycin-resistant E. faecium(including concurrent bacteremias) <br />for use in children and newborns against Gram-positive infections<br />for treatment of complicated skin and skin-structure infections including those due to MRSA (including Gram-positive bacterial diabetic foot infections (MRSA) without concomitant osteomyelitis) <br />the only approved agent for treatment of hospital-acquired MDR S.pneumoniaeinfectionsand is the first and only oral drug approved for the treatment of VRE infections. <br />Brickner, S.J.; Barbachyn, M.R.; Hutchinson, D.K.; Manninen, P.R. J. Med. Chem. 2008,51, 1981-1990<br />
    35. 35. Conclusions<br />29<br />Early 1993 - first synthesis<br />April 1995 - entered phase I trials<br />1996 - initiated phase II studies<br />January of 1998 - phase III trials began <br />April 18, 2000 - approved by the FDA <br />January of 2008 - has been used in an estimated 3 million patients<br />Brickner, S.J.; Barbachyn, M.R.; Hutchinson, D.K.; Manninen, P.R. J. Med. Chem. 2008,51, 1981-1990<br />

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