- Email: [email protected]
Case studies in current drug development: ‘glycylcyclines’
Case studies in current drug development: ‘glycylcyclines’ Phaik-Eng Sum Glycylcyclines represent a new class of tetracycline antibiotics with potent antibacterial activities against resistant pathogens. One of the glycylcyclines, Tygacil1, was selected for further development and has been approved by the FDA. It has an expanded broad-spectrum of antibacterial activity both in vitro and in vivo. It is active against a wide range of clinically relevant pathogens including Gram-positive, Gram-negative, atypical, and anaerobic bacteria and bacterial strains carrying either or both of the two major forms of tetracycline resistance (efflux and ribosomal protection). Most importantly, it is active against the multiply antibiotic resistant Gram-positive pathogenic bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Addresses Chemical and Screening Sciences Department, Wyeth Research, 401 North Middletown Road, Pearl River, NY10965, USA
Mechanism of action Tetracyclines are known to accumulate in bacteria preventing bacterial protein biosynthesis primarily via a disruption of the codon–anticodon interaction between tRNA and mRNA, so that binding of aminoacyl-tRNA to the ribosomal acceptor (A) site is prevented. Although the mechanism is not fully understood, inhibition is likely to result from strong binding of the tetracyclines with the 30S ribosomal subunit [3]. Studies designed to characterize the nature of the tetracycline-binding domain have revealed that when bound to bacterial ribosomes, tetracycline protects base A892 in 16s RNA from reactivity toward dimethyl sulfate and enhances reactivity towards bases U1052 and C1054. These results suggest that the 892–1054 region of 16sRNA contributes together with 30S ribosomal proteins to the antibiotic binding domain [9,10].
Corresponding author: Sum, Phaik-Eng ([email protected])
Current Opinion in Chemical Biology 2006, 10:374–379 This review comes from a themed issue on Next-generation therapeutics Edited by Clifton E Barry III and Alex Matter Available online 27th June 2006 1367-5931/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2006.06.009
Introduction The tetracyclines are a group of clinically useful, broadspectrum antibiotics discovered at Lederle in 1945. These agents have been important medical products for the past 60 years [1,2]. However, their widespread use, not just for therapeutic purposes but also in animal feed and for crop protection, has caused many genera of bacteria to develop resistance to this class of compounds. Bacterial resistance has become so widespread that many significant infections are becoming resistant to commonly used antibiotics [3,4,5]. New classes of antibiotics are needed to address this rapid emergence of bacterial resistance, and the discovery of glycylcyclines (a type of tetracycline) represents a significant advance in this area [6]. Three glycylcyclines, DMG-MINO, DMG-DMDOT and TBG-MINO, have been studied extensively [7,8]. TBG-MINO [also known as GAR-936, tigecycline (US adopted name), and tygacilTM] was selected for further development and was approved by the FDA on June 15, 2005 as an injectable antibiotic. This review covers recent progress in the development of glycylcyclines. Current Opinion in Chemical Biology 2006, 10:374–379
Further evidence for this interaction was obtained from the crystal structure of complexes of the Thermus thermophilius 30S ribosomal subunit with tetracycline, published in 2000 by Ramakrishnan’s group [11]. Two binding sites for tetracycline within the small ribosomal subunit were identified. The better-occupied site is located near the acceptor site (the A site) for aminoacylated tRNA between the head and the body of the 30S subunit, and the less-occupied site is at the interface between three RNA domains in the body of the subunit. In a separate report by Franceschi’s group, six binding sites for tetracycline were identified in the 30S subunit [12]. However, both findings indicated that it was the physical blockage of the A-site tRNA binding by tetracycline bound at the primary binding site that accounts for the inhibitory action of tetracycline. For the glycylcyclines, the mechanism by which protein synthesis is inhibited appears to be similar to that of the older tetracyclines. Both in vitro transcription and translation are inhibited by the addition of a glycylcycline [13] and these assays indicate that the binding of the glycylcyclines to the prokaryotic ribosome is 10–100 fold stronger than for tetracycline or minocycline [14]. This increased ribosomal binding is probably the primary factor for the excellent activity of these compounds against the ribosomal protection mechanism of tetracycline resistance.
Mechanisms of resistance There are three mechanisms of resistance to tetracyclines among bacterial pathogens: first, efflux of antibiotic mediated by resistance proteins located in the bacterial cytoplasmic membrane; second, ribosomal protection; www.sciencedirect.com
Case studies in current drug development: ‘Glycylcyclines’ Sum 375
and third, inactivation of tetracycline antibiotic. In the efflux mechanism, the intracellular tetracycline concentration remains too low for effective binding to ribosome, and is found in both Gram-negative and Gram-positive organisms [15,16]. The ribosomal protection mechanism is mediated by a protein that interacts with the ribosome, allowing protein synthesis even in the presence of tetracycline, and is more widespread in Gram-positive organisms. Although the molecular basis of this resistance mechanism is not fully understood, it might involve modification of ribosomal RNA or protein that affects binding of antibiotic to the 30S ribosomal subunit [17]. The third type is chemical alteration of the tetracycline molecule by a reaction in the cytoplasm that requires oxygen. This renders the drug inactive as an inhibitor of protein synthesis. The altered tetracycline then diffuses out of the cell. This mechanism of resistance is poorly characterized and may not be expressed in the natural habitat of most pathogens, especially because foci of infection in the human body are usually poorly aerated [18]. The mechanism by which glycylcyclines can overcome efflux-based tetracycline resistance has been investigated. The crystal structure of DMG-DMDOT in complex with Tet repressor class D, TetR(D), has been determined at 2.4 A˚ resolution. Steric hindrance and the charged glycylamido group appear to interfere with the conformational changes required for the mechanism of induction, and lead to decreased induction efficiency [19]. The majority of tetracycline resistance determinants are located on plasmids or transposons. These determinants have been grouped into classes defined by lack of crosshybridization under stringent conditions. Gram-negative organisms (e.g., E. coli) with efflux resistance genes have been assigned as tetA tetD, etc., and Gram-positive organisms (e.g., Staphylococcus aureus and Streptococcus spp.), have been assigned as tetK, tetL, etc. For ribosomal protection resistance genes, tetM, tetO, etc., have been assigned. All 26 letters have now been used to assign the known determinants. A nomenclature employing numerals has been recommended for future determinants and SB Levy’s group has assumed the responsibility to coordinate the assignment [20].
Strategies in discovery and lead optimization New tetracycline derivatives can be obtained through biotransformation [21], total synthesis or semi-synthetic pathways [22]. However, few total syntheses or biotransformation are efficient enough to be economically useful. Furthermore, few synthetic pathways are available to effect the desired transformations yet preserve the complicated stereochemical and functional integrities of the tetracycline. The semi-synthetic pathway remains the most viable option and has been demonstrated in the www.sciencedirect.com
development of doxycycline, minocycline and, recently, tigecycline [7,8]. During the course of our research to discover new tetracyclines with improved activity and pharmacokinetic properties, numerous synthetic methods were developed to obtain new semi-synthetic derivatives. Concomitantly, biological assays were developed to assist in understanding the mechanism of resistance and the structure–function relationships amongst the tetracyclines. These assays evaluated growth inhibition of tetracycline-sensitive and resistant organisms; binding to the bacterial ribosome; binding to repressor; induction assay to determine the uptake of tetracycline by E. coli; and in vitro inhibition of protein synthesis in a cell-free system [8,13,23]. The systematic analysis of new tetracycline analogs based on the above assays provided valuable information on particular modifications and led to the rational design of compounds with improved potency and pharmacological properties. Tetracyclines are amphipathic (Figure 1). Previous structure–function studies indicated that modifications of the hydrophilic domain are not tolerated, whereas alterations in the hydrophobic area can lead to compounds with enhanced antimicrobial activity. Accordingly, new tetracycline derivatives with modifications at positions 7, 8 and 9 were synthesized. A novel process was developed to prepare 8-halogenated tetracyclines, heretofore unknown among semi-synthetic derivatives. These halogen derivatives can provide access to a series of new derivatives with different substitutions at the 8-position via crosscoupling reactions [24]. Initially, many compounds with modifications at the 9-position (e.g., 9-amino-mino, 9-formamido-mino) (Figure 2) showed some improvement in activity against Gram-positive bacteria carrying the tetM determinant, but poor activity against Gram-negative bacteria and bacteria carrying the efflux determinants [6,25]. These findings suggested that substituents at the 9-position might be responsible for the improved activity against resistant Gram-positive pathogens. Because tetracyclines target an intracellular site, they must penetrate the bacterial cell wall. It is possible that compounds showing good activity against Gram-positive bacteria, but not against Gram-negative strains, are either pumped out or unable to penetrate the Gram-negative cell wall. Many derivatives with overall poor antibacterial activity still inhibit protein synthesis in cell-free in vitro translation/transcription assays, suggesting that those compounds might have a permeability issue. The attachment of a glycyl moiety at the 9-position (e.g., 9-glycylamido-mino, designed to enhance permeation while maintaining ribosome binding, led to the discovery of a series of new tetracycline derivatives referred to as Current Opinion in Chemical Biology 2006, 10:374–379
376 Next-generation therapeutics
Figure 1
activity. The introduction of a heteroatom in the alkylamino-group reduced the antimicrobial activity. Tigecycline was selected for development on the basis of the improved activity against clinical isolates of E. coli encoding the tetA or tetC determinants and against methicillinresistant staphylococcal clinical isolates [8,27].
In vitro and in vivo activity and pharmacological properties
Tetracycline structure.
‘glycylcyclines’ [6,25]. Efficient synthetic methods were developed and more than 300 glycylcycline analogs were synthesized via parallel synthesis for lead optimization and structure–activity relationships study. These include the N,N-dimethylglycylamido derivatives of 6-demethyl6-deoxytetracycline (DMG-DMDOT), and N,Ndimethyglycylamido-minocycline (DMG-MINO) [6,26] and tigecycline [8,27] (Figure 3). Several factors appear to be affecting the antimicrobial properties. The optimal size of a single alkyl group on the glycyl nitrogen appears to be tert-butyl, or small cycloalkyl unit. For dialkylamino glycyl compounds, the dimethylamino and pyrrolidino groups are superior. The basic nitrogen of the glycyl unit is essential; the bromo- and acylamino- intermediates are much less active. Changes in the spatial arrangement between the nitrogen and the carbonyl group of the glycyl substituent decreased the
Many glycylcyclines exhibit expanded broad-spectrum antibacterial activity both in vitro and in vivo. The unique feature of glycylcyclines (including tigecycline) is the ability to overcome both the ribosomal protection (tetM) and efflux (tetA, tetB, tetC, tetD, tetK, etc.) resistance mechanisms associated with tetracyclines. The selected drug candidate, tigecycline, is active against a wide range of clinically relevant pathogens including Gram-positive, Gram-negative, atypical, and anaerobic bacteria and bacterial strains carrying either or both of the two major forms of tetracycline resistance (efflux and ribosomal protection). Most importantly, it is active against the multiply antibiotic resistant Gram-positive pathogenic bacteria, especially methicillin-resistant Staphylococcus aureus (MRSA) (MIC 0.5 mg/ml), vancomycin-resistant enterococci (VRE) (MIC 0.25 mg/ml), and penicillin-resistant Streptococcus pneumoniae (PRSP) (MIC 0.12 mg/ml) and resistant Gram-negative organisms expressing an extended spectrum of b-lactamases [27–32,37]. In general, although tetracyclines are considered bacteriostatic, the in vitro studies have demonstrated that tigecycline is bactericidal against some bacteria strains [43]. The efficacy of tigecycline was demonstrated in a number of models: acute lethal infection in mouse, mouse thigh infection, mouse pneumonia model and a rat model of endocarditis. Good in vivo efficacy was observed with tigecycline and other glycylcycline derivatives
Figure 2
Tetracyclines substituted at the 9-position. Current Opinion in Chemical Biology 2006, 10:374–379
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Case studies in current drug development: ‘Glycylcyclines’ Sum 377
Figure 3
Some effective glycylcycline analogs.
(DMG-DMDOT, DMG-MINO) [26,27]. Utilizing systemic infections developed against the same strains used in the in vitro screening enabled direct comparison of activity and efficacy. When dosed intravenously, the glycylcyclines were as effective as minocycline against infections caused by minocycline-susceptible bacteria including MRSA and tetK-containing S. aureus. Infections caused by E. coli strains carrying tetA, tetB, tetC or tetM were also more responsive to treatment with tigecycline or DMGDMDOT than minocycline. Tigecycline, however, exceeded the activity of DMG-DMDOT against infections with the tetA- and tetC-containing strains, thus reflecting the improved in vitro activity of tigecycline over DMGDMDOT. Subsequent studies by a variety of investigators have confirmed the in vivo activity of tigecycline in a variety of animal models of infection [29,33–39]. In addition, tigecycline also exhibited potent activity against organisms causing community-acquired respiratory tract infection and nosocomial pneumonia [41]. Tigecycline has prolonged PAE and good tissue distribution and has a long half-life of about 30 hours in humans. To date, there are no drug-drug interactions, and no antagonism to other antibiotics [43].
Potential for rapid emergence of resistance Tigecycline is active against bacterial harboring in all of the tetracycline resistance genes tested. To address whether the common resistance genes can mutate and render cells resistant to the glycylcyclines, a series of experiments were performed including: www.sciencedirect.com
1. Direct selection for increased resistance. 2. Repeated passaging of strains harboring resistance genes. 3. In vitro and in vivo mutagenesis of conditionally expressed resistance genes. The principal findings of these investigations are that mutations giving rise to strains with decreased susceptibility to the glycylcyclines are difficult to obtain, and when they were obtained, only a four- to eight-fold increase in resistance was observed for DMG-DMDOT and DMG-MINO, but with a concomitant loss in resistance to tetracycline. Furthermore, the DMG-DMDOTresistant strains showed no increase in resistance to TBGMINO (t-butylaminoacetamido-minocycline). Although resistance to these compounds will certainly arise if they are used extensively, it might not occur as the result of straightforward alteration in the existing resistance genes. Limited cross-resistance has been observed between tigecycline and other antibiotics [44–46].
Conclusions The development of tigecycline (Tygacil1) represents an important milestone in addressing the rapid emergence of bacterial resistance to tetracyclines and other currently available antibiotics. For patients with serious infection, the initial choice for empirical therapy with broad-spectrum antibiotics is crucial. Tygacil can be used as an empiric monotherapy to treat a variety of complicated intra-abdominal infections and complicated skin and skin structure infections, both hospital-acquired and Current Opinion in Chemical Biology 2006, 10:374–379
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community-acquired, including complicated appendicitis, infected burns, intra-abdominal abscesses, deep soft tissue infections, and infected ulcers [35,40–42,47]. It also provides clinicians with a novel, broad-spectrum option that can be used at the onset of treatment when the specific bacteria present are not yet known. It is very likely that, with time, bacterial resistance to Tygacil will be more frequent and again raise concerns among medical professionals. It is hoped that the total synthesis route will provide access to analogs that are otherwise unobtainable from biotransformation, and that the X-ray structure of the tetracycline–ribosome complex will provide some valuable information in assisting the design of the next generation of novel tetracycline derivatives to combat any future emergence of bacterial resistance.
Acknowledgements The author thanks Steve Tam, Tarek Mansour, and Alan G Sutherland for helpful comments.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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Chopra I, Roberts M: Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol Mol Biol Rev 2001, 65:232-260.
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Chopra I: Antibiotic resistance in Staphylococcus aureus: concerns, causes and cures. Expert Rev Anti Infect Ther 2003, 1:45-55.
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Cookson B: Clinical significance of emergence of bacterial antimicrobial resistance in the hospital environment. J Appl Microbiol 2005, 99:989-996. The paper provides updated information on the emergence of antibiotic resistance in the hospital environment. 6.
Sum PE, Lee VJ, Testa RT, Hlavka JJ, Ellestad GA, Bloom JD, Gluzman Y, Tally FP: Glycylcyclines 1. a new generation of potent antibacterial agents through modification of 9-aminotetracyclines. J Med Chem 1994, 37:184-188.
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Sum PE, Sum FW, Projan SJ: Recent developments in tetracycline antibiotics. Curr Pharm Des 1998, 4:119-132.
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Sum PE, Petersen PJ: Synthesis and structure-activity relationship of novel glycylcycline derivatives leading to the discovery of GAR-936. Bioorg Med Chem Lett 1999, 9:1459-1462.
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Buck MA, Cooperman BS: Single protein omission reconstitution studies of tetracycline binding to the 30S subunit of Escherichia coli ribosomes. Biochemistry 1990, 29:5374-5379.
10. Bauer G, Berens C, Projan SJ, Hillen W: Comparison of tetracycline and tigecycline binding to ribosomes mapped by dimethylsulphate and drug-directed Fe2+ cleavage of 16S rRNA. J Antimicrob Chemother 2004, 53:592-599. Current Opinion in Chemical Biology 2006, 10:374–379
11. Brodersen DE, Clemons WM Jr, Carter AP, Morgan-Warren RJ, Wimberly BT, Ramakrishnan V: The structure basis for the action of the antibiotics tetracycline, pactamycin, and hygromycin B on the 30S ribosomal subunit. Cell 2000, 103:1143-1154. 12. Pioletti M, Schlunzen F, Harms J, Zarivach R, Gluhmann M, Avila H, Bashan A, Bartels H, Auerbach T, Jacobi C et al.: Crystal structure of complexes of the small ribosomal subunit with tetracycline, edeine and IF3. EMBO J 2001, 20:1829. 13. Rasmussen BA, Gluzman Y, Tally FP: Inhibition of protein synthesis on tetracycline-resistant TetM-protected ribosomes by a novel class of tetracyclines, the glycylcyclines. Antimicrob Agents Chemother 1994, 38:1658-1660. 14. Bergeron J, Ammirati M, Danley D, Norcia M, Retsema J, Strick CA, Su WG, Sutcliffe J, Wondrack L: Glycylcyclines bind to the high-affinity tetracycline ribosomal binding site and evade Tet(M) and Tet(O)-mediated ribosomal protection. Antimicrob Agents Chemother 1996, 40:2226-2228. 15. Salyers AA, Speer BS, Shoemaker NB: New perspective in tetracycline resistance. Mol Microbiol 1990, 4:151-156. 16. Sapunaric FM, Aldema-Ramos M, McMurry LM: Tetracycline resistance: efflux, mutation, and other mechanisms. In Frontiers In Antimicrobial Resistance. Edited by White DG, Alekshun MN, McDermott PF. American Society for Microbiology; 2005:3-18. A good update on bacterial mechanisms of resistance, especially on efflux, inactivation and rRNA mutations. 17. Roberts MC: Tetracycline resistance due to ribosomal protection proteins. In Frontiers Antimicrobial Resistance. Edited by White DG, Alekshun MN, McDermott PF. American Society for Microbiology; 2005:19-28. 18. Moore IF, Donald WH, Gerard DW: Tigecycline is modified by the flavin-dependent monooxygenase TetX. Biochemistry 2005, 44:11829-11835. 19. Orth P, Schnappinger D, Sum PE, Ellestad GA, Hillen W, Saenger W, Hinrichs W: Crystal structure of the Tet repressor in complex with a novel tetracycline, 9-(N,Ndimethylglycylamido)-6-demethyl-6-deoxy-tetracycline. J Mol Biol 1999, 285:455-461. 20. Levy SB, McMurry LM, Barbosa TM, Burdett V, Courvalin P, Hillen W, Roberts MC, Rood JI, Taylor DE: Nomenclature for new tetracycline resistance determinants. Antimicrob Agents Chemother 1999, 43:1523-1524. 21. Khosla C, Tang Y: Chemistry: a new route to designer antibiotics. Science 2005, 308:367-368. 22. Charest MG, Lerner CD, Brubaker JD, Siegel DR, Myers AG: A convergent enantioselective route to structurally diverse 6 - deoxytetracycline antibiotics. Science 2005, 308:395-398. This paper describes the first efficient total synthesis of an intact tetracycline molecule. The method provides accessibility to analogs that could not be obtained via fermentation or semi-synthetic pathways. A number of new analogs with modification of the D-ring were synthesized, and minimum inhibitory concentrations were obtained for those compounds in a panel of Gram-positive and Gram-negative bacteria. 23. Lederer T, Kintrup M, Takahashi M, Sum PE, Ellestad GA, Hillen W: Tetracycline analogs affecting binding to Tn10-encoded Tet repressor trigger the same mechanism of induction. Biochemistry 1996, 35:7439-7446. 24. Sum PE, Lee VJ, Tally FP: Synthesis of novel tetracycline derivatives with substitution at the C-8 position. Tetrahedon Lett 1994, 35:1835-1836. 25. Sum PE, Ross AT, Petersen PJ, Testa RT: Synthesis and antibacterial activity of 9-substituted minocycline derivatives. Bioorg Med Chem Lett 2006, 16:400-403. 26. Testa RT, Petersen PJ, Jacobus NV, Sum PE, Lee VJ, Tally FP: In vitro and in vivo antibacterial activities of the glycylcyclines, a new class of semisynthetic tetracyclines. Antimicrob Agents Chemother 1993, 37:2270-2277. 27. Petersen PJ, Jacobus NV, Weiss WJ, Sum PE, Testa RT: In vitro and In vivo antimicrobial activities of a novel glycylcycline, the www.sciencedirect.com
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9-t-butylglycylamido derivative of minocycline (GAR-936). Antimicrob Agents Chemother 1999, 43:738-744. 28. Betriu C, Culebras E, Rodriguez-Avial I, Gomez M, Sanchez BA, Picazo JJ: In vitro activities of tigecycline against erythromycin-resistant Streptococcus pyogenes and Streptococcus agalactiae: mechanisms of macrolide and tetracycline resistance. Antimicrob Agents Chemother 2004, 48:323-325. 29. Cercenado E, Cercenado S, Bouza E: In vitro activities of tigecycline (GAR-936) and 12 other antimicrobial agents against 90 eikenella corrodens clinical isolates. Antimicrob Agents Chemother 2003, 47:2644-2645. 30. Edelstein PH, Weiss WJ, Edelstein MAC: Activities of tigecycline (GAR-936) against Legionella pneumophila in vitro and in guinea pigs with L. pneumophila pneumonia. Antimicrob Agents Chemother 2003, 47:533-540. 31. Jacobus NV, McDermott LA, Ruthazer R, Snydman DR: In vitro activities of tigecycline against the Bacteroides fragilis group. Antimicrob Agents Chemother 2004, 48:1034-1036. 32. Kitzis MD, Ly A, Goldstein FW: In vitro activities of tigecycline (GAR-936) against multidrug-resistant staphylococcus aureus and streptococcus pneumoniae. Antimicrob Agents Chemother 2004, 48:366-367.
38. Lefort A, Lafaurie M, Massias L, Petegnief Y, Saleh-Mghir A, Muller-Serieys C, Le Guludec D, Fantin B: Activity and diffusion of tigecycline (GAR-936) in experimental enterococcal endocarditis. Antimicrob Agents Chemother 2003, 47:216-222. 39. Van Ogtrop ML, Andes D, Stamstad TJ, Conklin B, Weiss WJ, Craig WA, Vesga O: In vivo pharmacodynamic activities of two glycylcyclines (GAR-936 and WAY 152,288) against various Gram-positive and Gram-negative bacteria. Antimicrob Agents Chemother 2000, 44:943-949. 40. Fritsche TR, Sader HS, Stilwell MG, Dowzicky MJ, Jones RN: Potency and spectrum of tigecycline tested against an international collection of bacterial pathogens associated with skin and soft tissue infections (2000–2004). Diagn Microbiol Infect Dis 2005, 52:195-201. 41. Fritsche TR, Sader HS, Stilwell MG, Dowzicky MJ, Jones RN: Antimicrobial activity of tigecycline tested against organisms causing community-acquired respiratory tract infection and nosocomial pneumonia. Diagn Microbiol Infect Dis 2005, 52:187-193. 42. Fritsche TR, Strabala PA, Sader HS, Dowzicky MJ, Jones RN: Activity of tigecycline tested against a global collection of enterobacteriaceae, including tetracycline-resistant isolates. Diagn Microbiol Infect Dis 2005, 52:209-213.
33. Nannini EC, Pai SR, Singh KV, Murray BE: Activity of tigecycline (GAR-936), a novel glycylcycline, against enterococci in the mouse peritonitis model. Antimicrob Agents Chemother 2003, 47:529-532.
43. Projan SJ: Preclinical pharmacology of GAR-936, a novel glycylcycline antibacterial agent. Pharmacotherapy 2000, 20:219S-223S.
34. Pachon-Ibanez ME, Jimenez-Mejias ME, Pichardo C, Llanos AC, Pachon J: Activity of tigecycline (GAR-936) against Acinetobacter baumannii strains, including those resistant to imipenem. Antimicrob Agents Chemother 2004, 48:4479-4481.
44. Dean CR, Visalli MA, Projan SJ, Sum PE, Bradford PA: Efflux-mediated resistance to tigecycline (GAR-936) in Pseudomonas aeruginosa PAO1. Antimicrob Agents Chemother 2003, 47:972-978.
35. Roberts MC: Tetracycline therapy: update. Clin Infect Dis 2003, 36:462-467.
45. Ruzin A, Visalli MA, Keeney D, Bradford PA: Influence of transcriptional activator RamA on expression of multidrug efflux pump AcrAB and tigecycline susceptibility in Klebsiella pneumoniae. Antimicrob Agents Chemother 2005, 49:1017-1022.
36. Yin LY, Lazzarini L, Li F, Stevens CM, Calhoun JH: Comparative evaluation of tigecycline and vancomycin, with and without rifampicin, in the treatment of methicillin-resistant Staphylococcus aureus experimental osteomyelitis in a rabbit model. J Antimicrob Chemother 2005, 55:995-1002. 37. Petersen PJ, Bradford PA, Weiss WJ, Murphy TM, Sum PE, Projan SJ: In vitro and in vivo activities of tigecycline (GAR936), daptomycin, and comparative antimicrobial agents against glycopeptide-intermediate Staphylococcus aureus and other resistant Gram-positive pathogens. Antimicrob Agents Chemother 2002, 46:2595-2601.
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46. Ruzin A, Keeney D, Bradford PA: AcrAB efflux pump plays a role in decreased susceptibility to tigecycline in Morganella morganii. Antimicrob Agents Chemother 2005, 49:791-793. 47. Wenzel R, Bate G, Kirkpatrick P: Fresh from the pipeline: tigecycline. Nat Rev Drug Discov 2005, 4:809-810. This paper provides a nice update on clinical data and indications. It also gives an updated market analysis for intravenous antibiotics.
Current Opinion in Chemical Biology 2006, 10:374–379
Mechanism of action Tetracyclines are known to accumulate in bacteria preventing bacterial protein biosynthesis primarily via a disruption of the codon–anticodon interaction between tRNA and mRNA, so that binding of aminoacyl-tRNA to the ribosomal acceptor (A) site is prevented. Although the mechanism is not fully understood, inhibition is likely to result from strong binding of the tetracyclines with the 30S ribosomal subunit [3]. Studies designed to characterize the nature of the tetracycline-binding domain have revealed that when bound to bacterial ribosomes, tetracycline protects base A892 in 16s RNA from reactivity toward dimethyl sulfate and enhances reactivity towards bases U1052 and C1054. These results suggest that the 892–1054 region of 16sRNA contributes together with 30S ribosomal proteins to the antibiotic binding domain [9,10].
Corresponding author: Sum, Phaik-Eng ([email protected])
Current Opinion in Chemical Biology 2006, 10:374–379 This review comes from a themed issue on Next-generation therapeutics Edited by Clifton E Barry III and Alex Matter Available online 27th June 2006 1367-5931/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2006.06.009
Introduction The tetracyclines are a group of clinically useful, broadspectrum antibiotics discovered at Lederle in 1945. These agents have been important medical products for the past 60 years [1,2]. However, their widespread use, not just for therapeutic purposes but also in animal feed and for crop protection, has caused many genera of bacteria to develop resistance to this class of compounds. Bacterial resistance has become so widespread that many significant infections are becoming resistant to commonly used antibiotics [3,4,5]. New classes of antibiotics are needed to address this rapid emergence of bacterial resistance, and the discovery of glycylcyclines (a type of tetracycline) represents a significant advance in this area [6]. Three glycylcyclines, DMG-MINO, DMG-DMDOT and TBG-MINO, have been studied extensively [7,8]. TBG-MINO [also known as GAR-936, tigecycline (US adopted name), and tygacilTM] was selected for further development and was approved by the FDA on June 15, 2005 as an injectable antibiotic. This review covers recent progress in the development of glycylcyclines. Current Opinion in Chemical Biology 2006, 10:374–379
Further evidence for this interaction was obtained from the crystal structure of complexes of the Thermus thermophilius 30S ribosomal subunit with tetracycline, published in 2000 by Ramakrishnan’s group [11]. Two binding sites for tetracycline within the small ribosomal subunit were identified. The better-occupied site is located near the acceptor site (the A site) for aminoacylated tRNA between the head and the body of the 30S subunit, and the less-occupied site is at the interface between three RNA domains in the body of the subunit. In a separate report by Franceschi’s group, six binding sites for tetracycline were identified in the 30S subunit [12]. However, both findings indicated that it was the physical blockage of the A-site tRNA binding by tetracycline bound at the primary binding site that accounts for the inhibitory action of tetracycline. For the glycylcyclines, the mechanism by which protein synthesis is inhibited appears to be similar to that of the older tetracyclines. Both in vitro transcription and translation are inhibited by the addition of a glycylcycline [13] and these assays indicate that the binding of the glycylcyclines to the prokaryotic ribosome is 10–100 fold stronger than for tetracycline or minocycline [14]. This increased ribosomal binding is probably the primary factor for the excellent activity of these compounds against the ribosomal protection mechanism of tetracycline resistance.
Mechanisms of resistance There are three mechanisms of resistance to tetracyclines among bacterial pathogens: first, efflux of antibiotic mediated by resistance proteins located in the bacterial cytoplasmic membrane; second, ribosomal protection; www.sciencedirect.com
Case studies in current drug development: ‘Glycylcyclines’ Sum 375
and third, inactivation of tetracycline antibiotic. In the efflux mechanism, the intracellular tetracycline concentration remains too low for effective binding to ribosome, and is found in both Gram-negative and Gram-positive organisms [15,16]. The ribosomal protection mechanism is mediated by a protein that interacts with the ribosome, allowing protein synthesis even in the presence of tetracycline, and is more widespread in Gram-positive organisms. Although the molecular basis of this resistance mechanism is not fully understood, it might involve modification of ribosomal RNA or protein that affects binding of antibiotic to the 30S ribosomal subunit [17]. The third type is chemical alteration of the tetracycline molecule by a reaction in the cytoplasm that requires oxygen. This renders the drug inactive as an inhibitor of protein synthesis. The altered tetracycline then diffuses out of the cell. This mechanism of resistance is poorly characterized and may not be expressed in the natural habitat of most pathogens, especially because foci of infection in the human body are usually poorly aerated [18]. The mechanism by which glycylcyclines can overcome efflux-based tetracycline resistance has been investigated. The crystal structure of DMG-DMDOT in complex with Tet repressor class D, TetR(D), has been determined at 2.4 A˚ resolution. Steric hindrance and the charged glycylamido group appear to interfere with the conformational changes required for the mechanism of induction, and lead to decreased induction efficiency [19]. The majority of tetracycline resistance determinants are located on plasmids or transposons. These determinants have been grouped into classes defined by lack of crosshybridization under stringent conditions. Gram-negative organisms (e.g., E. coli) with efflux resistance genes have been assigned as tetA tetD, etc., and Gram-positive organisms (e.g., Staphylococcus aureus and Streptococcus spp.), have been assigned as tetK, tetL, etc. For ribosomal protection resistance genes, tetM, tetO, etc., have been assigned. All 26 letters have now been used to assign the known determinants. A nomenclature employing numerals has been recommended for future determinants and SB Levy’s group has assumed the responsibility to coordinate the assignment [20].
Strategies in discovery and lead optimization New tetracycline derivatives can be obtained through biotransformation [21], total synthesis or semi-synthetic pathways [22]. However, few total syntheses or biotransformation are efficient enough to be economically useful. Furthermore, few synthetic pathways are available to effect the desired transformations yet preserve the complicated stereochemical and functional integrities of the tetracycline. The semi-synthetic pathway remains the most viable option and has been demonstrated in the www.sciencedirect.com
development of doxycycline, minocycline and, recently, tigecycline [7,8]. During the course of our research to discover new tetracyclines with improved activity and pharmacokinetic properties, numerous synthetic methods were developed to obtain new semi-synthetic derivatives. Concomitantly, biological assays were developed to assist in understanding the mechanism of resistance and the structure–function relationships amongst the tetracyclines. These assays evaluated growth inhibition of tetracycline-sensitive and resistant organisms; binding to the bacterial ribosome; binding to repressor; induction assay to determine the uptake of tetracycline by E. coli; and in vitro inhibition of protein synthesis in a cell-free system [8,13,23]. The systematic analysis of new tetracycline analogs based on the above assays provided valuable information on particular modifications and led to the rational design of compounds with improved potency and pharmacological properties. Tetracyclines are amphipathic (Figure 1). Previous structure–function studies indicated that modifications of the hydrophilic domain are not tolerated, whereas alterations in the hydrophobic area can lead to compounds with enhanced antimicrobial activity. Accordingly, new tetracycline derivatives with modifications at positions 7, 8 and 9 were synthesized. A novel process was developed to prepare 8-halogenated tetracyclines, heretofore unknown among semi-synthetic derivatives. These halogen derivatives can provide access to a series of new derivatives with different substitutions at the 8-position via crosscoupling reactions [24]. Initially, many compounds with modifications at the 9-position (e.g., 9-amino-mino, 9-formamido-mino) (Figure 2) showed some improvement in activity against Gram-positive bacteria carrying the tetM determinant, but poor activity against Gram-negative bacteria and bacteria carrying the efflux determinants [6,25]. These findings suggested that substituents at the 9-position might be responsible for the improved activity against resistant Gram-positive pathogens. Because tetracyclines target an intracellular site, they must penetrate the bacterial cell wall. It is possible that compounds showing good activity against Gram-positive bacteria, but not against Gram-negative strains, are either pumped out or unable to penetrate the Gram-negative cell wall. Many derivatives with overall poor antibacterial activity still inhibit protein synthesis in cell-free in vitro translation/transcription assays, suggesting that those compounds might have a permeability issue. The attachment of a glycyl moiety at the 9-position (e.g., 9-glycylamido-mino, designed to enhance permeation while maintaining ribosome binding, led to the discovery of a series of new tetracycline derivatives referred to as Current Opinion in Chemical Biology 2006, 10:374–379
376 Next-generation therapeutics
Figure 1
activity. The introduction of a heteroatom in the alkylamino-group reduced the antimicrobial activity. Tigecycline was selected for development on the basis of the improved activity against clinical isolates of E. coli encoding the tetA or tetC determinants and against methicillinresistant staphylococcal clinical isolates [8,27].
In vitro and in vivo activity and pharmacological properties
Tetracycline structure.
‘glycylcyclines’ [6,25]. Efficient synthetic methods were developed and more than 300 glycylcycline analogs were synthesized via parallel synthesis for lead optimization and structure–activity relationships study. These include the N,N-dimethylglycylamido derivatives of 6-demethyl6-deoxytetracycline (DMG-DMDOT), and N,Ndimethyglycylamido-minocycline (DMG-MINO) [6,26] and tigecycline [8,27] (Figure 3). Several factors appear to be affecting the antimicrobial properties. The optimal size of a single alkyl group on the glycyl nitrogen appears to be tert-butyl, or small cycloalkyl unit. For dialkylamino glycyl compounds, the dimethylamino and pyrrolidino groups are superior. The basic nitrogen of the glycyl unit is essential; the bromo- and acylamino- intermediates are much less active. Changes in the spatial arrangement between the nitrogen and the carbonyl group of the glycyl substituent decreased the
Many glycylcyclines exhibit expanded broad-spectrum antibacterial activity both in vitro and in vivo. The unique feature of glycylcyclines (including tigecycline) is the ability to overcome both the ribosomal protection (tetM) and efflux (tetA, tetB, tetC, tetD, tetK, etc.) resistance mechanisms associated with tetracyclines. The selected drug candidate, tigecycline, is active against a wide range of clinically relevant pathogens including Gram-positive, Gram-negative, atypical, and anaerobic bacteria and bacterial strains carrying either or both of the two major forms of tetracycline resistance (efflux and ribosomal protection). Most importantly, it is active against the multiply antibiotic resistant Gram-positive pathogenic bacteria, especially methicillin-resistant Staphylococcus aureus (MRSA) (MIC 0.5 mg/ml), vancomycin-resistant enterococci (VRE) (MIC 0.25 mg/ml), and penicillin-resistant Streptococcus pneumoniae (PRSP) (MIC 0.12 mg/ml) and resistant Gram-negative organisms expressing an extended spectrum of b-lactamases [27–32,37]. In general, although tetracyclines are considered bacteriostatic, the in vitro studies have demonstrated that tigecycline is bactericidal against some bacteria strains [43]. The efficacy of tigecycline was demonstrated in a number of models: acute lethal infection in mouse, mouse thigh infection, mouse pneumonia model and a rat model of endocarditis. Good in vivo efficacy was observed with tigecycline and other glycylcycline derivatives
Figure 2
Tetracyclines substituted at the 9-position. Current Opinion in Chemical Biology 2006, 10:374–379
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Case studies in current drug development: ‘Glycylcyclines’ Sum 377
Figure 3
Some effective glycylcycline analogs.
(DMG-DMDOT, DMG-MINO) [26,27]. Utilizing systemic infections developed against the same strains used in the in vitro screening enabled direct comparison of activity and efficacy. When dosed intravenously, the glycylcyclines were as effective as minocycline against infections caused by minocycline-susceptible bacteria including MRSA and tetK-containing S. aureus. Infections caused by E. coli strains carrying tetA, tetB, tetC or tetM were also more responsive to treatment with tigecycline or DMGDMDOT than minocycline. Tigecycline, however, exceeded the activity of DMG-DMDOT against infections with the tetA- and tetC-containing strains, thus reflecting the improved in vitro activity of tigecycline over DMGDMDOT. Subsequent studies by a variety of investigators have confirmed the in vivo activity of tigecycline in a variety of animal models of infection [29,33–39]. In addition, tigecycline also exhibited potent activity against organisms causing community-acquired respiratory tract infection and nosocomial pneumonia [41]. Tigecycline has prolonged PAE and good tissue distribution and has a long half-life of about 30 hours in humans. To date, there are no drug-drug interactions, and no antagonism to other antibiotics [43].
Potential for rapid emergence of resistance Tigecycline is active against bacterial harboring in all of the tetracycline resistance genes tested. To address whether the common resistance genes can mutate and render cells resistant to the glycylcyclines, a series of experiments were performed including: www.sciencedirect.com
1. Direct selection for increased resistance. 2. Repeated passaging of strains harboring resistance genes. 3. In vitro and in vivo mutagenesis of conditionally expressed resistance genes. The principal findings of these investigations are that mutations giving rise to strains with decreased susceptibility to the glycylcyclines are difficult to obtain, and when they were obtained, only a four- to eight-fold increase in resistance was observed for DMG-DMDOT and DMG-MINO, but with a concomitant loss in resistance to tetracycline. Furthermore, the DMG-DMDOTresistant strains showed no increase in resistance to TBGMINO (t-butylaminoacetamido-minocycline). Although resistance to these compounds will certainly arise if they are used extensively, it might not occur as the result of straightforward alteration in the existing resistance genes. Limited cross-resistance has been observed between tigecycline and other antibiotics [44–46].
Conclusions The development of tigecycline (Tygacil1) represents an important milestone in addressing the rapid emergence of bacterial resistance to tetracyclines and other currently available antibiotics. For patients with serious infection, the initial choice for empirical therapy with broad-spectrum antibiotics is crucial. Tygacil can be used as an empiric monotherapy to treat a variety of complicated intra-abdominal infections and complicated skin and skin structure infections, both hospital-acquired and Current Opinion in Chemical Biology 2006, 10:374–379
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community-acquired, including complicated appendicitis, infected burns, intra-abdominal abscesses, deep soft tissue infections, and infected ulcers [35,40–42,47]. It also provides clinicians with a novel, broad-spectrum option that can be used at the onset of treatment when the specific bacteria present are not yet known. It is very likely that, with time, bacterial resistance to Tygacil will be more frequent and again raise concerns among medical professionals. It is hoped that the total synthesis route will provide access to analogs that are otherwise unobtainable from biotransformation, and that the X-ray structure of the tetracycline–ribosome complex will provide some valuable information in assisting the design of the next generation of novel tetracycline derivatives to combat any future emergence of bacterial resistance.
Acknowledgements The author thanks Steve Tam, Tarek Mansour, and Alan G Sutherland for helpful comments.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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11. Brodersen DE, Clemons WM Jr, Carter AP, Morgan-Warren RJ, Wimberly BT, Ramakrishnan V: The structure basis for the action of the antibiotics tetracycline, pactamycin, and hygromycin B on the 30S ribosomal subunit. Cell 2000, 103:1143-1154. 12. Pioletti M, Schlunzen F, Harms J, Zarivach R, Gluhmann M, Avila H, Bashan A, Bartels H, Auerbach T, Jacobi C et al.: Crystal structure of complexes of the small ribosomal subunit with tetracycline, edeine and IF3. EMBO J 2001, 20:1829. 13. Rasmussen BA, Gluzman Y, Tally FP: Inhibition of protein synthesis on tetracycline-resistant TetM-protected ribosomes by a novel class of tetracyclines, the glycylcyclines. Antimicrob Agents Chemother 1994, 38:1658-1660. 14. Bergeron J, Ammirati M, Danley D, Norcia M, Retsema J, Strick CA, Su WG, Sutcliffe J, Wondrack L: Glycylcyclines bind to the high-affinity tetracycline ribosomal binding site and evade Tet(M) and Tet(O)-mediated ribosomal protection. Antimicrob Agents Chemother 1996, 40:2226-2228. 15. Salyers AA, Speer BS, Shoemaker NB: New perspective in tetracycline resistance. Mol Microbiol 1990, 4:151-156. 16. Sapunaric FM, Aldema-Ramos M, McMurry LM: Tetracycline resistance: efflux, mutation, and other mechanisms. In Frontiers In Antimicrobial Resistance. Edited by White DG, Alekshun MN, McDermott PF. American Society for Microbiology; 2005:3-18. A good update on bacterial mechanisms of resistance, especially on efflux, inactivation and rRNA mutations. 17. Roberts MC: Tetracycline resistance due to ribosomal protection proteins. In Frontiers Antimicrobial Resistance. Edited by White DG, Alekshun MN, McDermott PF. American Society for Microbiology; 2005:19-28. 18. Moore IF, Donald WH, Gerard DW: Tigecycline is modified by the flavin-dependent monooxygenase TetX. Biochemistry 2005, 44:11829-11835. 19. Orth P, Schnappinger D, Sum PE, Ellestad GA, Hillen W, Saenger W, Hinrichs W: Crystal structure of the Tet repressor in complex with a novel tetracycline, 9-(N,Ndimethylglycylamido)-6-demethyl-6-deoxy-tetracycline. J Mol Biol 1999, 285:455-461. 20. Levy SB, McMurry LM, Barbosa TM, Burdett V, Courvalin P, Hillen W, Roberts MC, Rood JI, Taylor DE: Nomenclature for new tetracycline resistance determinants. Antimicrob Agents Chemother 1999, 43:1523-1524. 21. Khosla C, Tang Y: Chemistry: a new route to designer antibiotics. Science 2005, 308:367-368. 22. Charest MG, Lerner CD, Brubaker JD, Siegel DR, Myers AG: A convergent enantioselective route to structurally diverse 6 - deoxytetracycline antibiotics. Science 2005, 308:395-398. This paper describes the first efficient total synthesis of an intact tetracycline molecule. The method provides accessibility to analogs that could not be obtained via fermentation or semi-synthetic pathways. A number of new analogs with modification of the D-ring were synthesized, and minimum inhibitory concentrations were obtained for those compounds in a panel of Gram-positive and Gram-negative bacteria. 23. Lederer T, Kintrup M, Takahashi M, Sum PE, Ellestad GA, Hillen W: Tetracycline analogs affecting binding to Tn10-encoded Tet repressor trigger the same mechanism of induction. Biochemistry 1996, 35:7439-7446. 24. Sum PE, Lee VJ, Tally FP: Synthesis of novel tetracycline derivatives with substitution at the C-8 position. Tetrahedon Lett 1994, 35:1835-1836. 25. Sum PE, Ross AT, Petersen PJ, Testa RT: Synthesis and antibacterial activity of 9-substituted minocycline derivatives. Bioorg Med Chem Lett 2006, 16:400-403. 26. Testa RT, Petersen PJ, Jacobus NV, Sum PE, Lee VJ, Tally FP: In vitro and in vivo antibacterial activities of the glycylcyclines, a new class of semisynthetic tetracyclines. Antimicrob Agents Chemother 1993, 37:2270-2277. 27. Petersen PJ, Jacobus NV, Weiss WJ, Sum PE, Testa RT: In vitro and In vivo antimicrobial activities of a novel glycylcycline, the www.sciencedirect.com
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9-t-butylglycylamido derivative of minocycline (GAR-936). Antimicrob Agents Chemother 1999, 43:738-744. 28. Betriu C, Culebras E, Rodriguez-Avial I, Gomez M, Sanchez BA, Picazo JJ: In vitro activities of tigecycline against erythromycin-resistant Streptococcus pyogenes and Streptococcus agalactiae: mechanisms of macrolide and tetracycline resistance. Antimicrob Agents Chemother 2004, 48:323-325. 29. Cercenado E, Cercenado S, Bouza E: In vitro activities of tigecycline (GAR-936) and 12 other antimicrobial agents against 90 eikenella corrodens clinical isolates. Antimicrob Agents Chemother 2003, 47:2644-2645. 30. Edelstein PH, Weiss WJ, Edelstein MAC: Activities of tigecycline (GAR-936) against Legionella pneumophila in vitro and in guinea pigs with L. pneumophila pneumonia. Antimicrob Agents Chemother 2003, 47:533-540. 31. Jacobus NV, McDermott LA, Ruthazer R, Snydman DR: In vitro activities of tigecycline against the Bacteroides fragilis group. Antimicrob Agents Chemother 2004, 48:1034-1036. 32. Kitzis MD, Ly A, Goldstein FW: In vitro activities of tigecycline (GAR-936) against multidrug-resistant staphylococcus aureus and streptococcus pneumoniae. Antimicrob Agents Chemother 2004, 48:366-367.
38. Lefort A, Lafaurie M, Massias L, Petegnief Y, Saleh-Mghir A, Muller-Serieys C, Le Guludec D, Fantin B: Activity and diffusion of tigecycline (GAR-936) in experimental enterococcal endocarditis. Antimicrob Agents Chemother 2003, 47:216-222. 39. Van Ogtrop ML, Andes D, Stamstad TJ, Conklin B, Weiss WJ, Craig WA, Vesga O: In vivo pharmacodynamic activities of two glycylcyclines (GAR-936 and WAY 152,288) against various Gram-positive and Gram-negative bacteria. Antimicrob Agents Chemother 2000, 44:943-949. 40. Fritsche TR, Sader HS, Stilwell MG, Dowzicky MJ, Jones RN: Potency and spectrum of tigecycline tested against an international collection of bacterial pathogens associated with skin and soft tissue infections (2000–2004). Diagn Microbiol Infect Dis 2005, 52:195-201. 41. Fritsche TR, Sader HS, Stilwell MG, Dowzicky MJ, Jones RN: Antimicrobial activity of tigecycline tested against organisms causing community-acquired respiratory tract infection and nosocomial pneumonia. Diagn Microbiol Infect Dis 2005, 52:187-193. 42. Fritsche TR, Strabala PA, Sader HS, Dowzicky MJ, Jones RN: Activity of tigecycline tested against a global collection of enterobacteriaceae, including tetracycline-resistant isolates. Diagn Microbiol Infect Dis 2005, 52:209-213.
33. Nannini EC, Pai SR, Singh KV, Murray BE: Activity of tigecycline (GAR-936), a novel glycylcycline, against enterococci in the mouse peritonitis model. Antimicrob Agents Chemother 2003, 47:529-532.
43. Projan SJ: Preclinical pharmacology of GAR-936, a novel glycylcycline antibacterial agent. Pharmacotherapy 2000, 20:219S-223S.
34. Pachon-Ibanez ME, Jimenez-Mejias ME, Pichardo C, Llanos AC, Pachon J: Activity of tigecycline (GAR-936) against Acinetobacter baumannii strains, including those resistant to imipenem. Antimicrob Agents Chemother 2004, 48:4479-4481.
44. Dean CR, Visalli MA, Projan SJ, Sum PE, Bradford PA: Efflux-mediated resistance to tigecycline (GAR-936) in Pseudomonas aeruginosa PAO1. Antimicrob Agents Chemother 2003, 47:972-978.
35. Roberts MC: Tetracycline therapy: update. Clin Infect Dis 2003, 36:462-467.
45. Ruzin A, Visalli MA, Keeney D, Bradford PA: Influence of transcriptional activator RamA on expression of multidrug efflux pump AcrAB and tigecycline susceptibility in Klebsiella pneumoniae. Antimicrob Agents Chemother 2005, 49:1017-1022.
36. Yin LY, Lazzarini L, Li F, Stevens CM, Calhoun JH: Comparative evaluation of tigecycline and vancomycin, with and without rifampicin, in the treatment of methicillin-resistant Staphylococcus aureus experimental osteomyelitis in a rabbit model. J Antimicrob Chemother 2005, 55:995-1002. 37. Petersen PJ, Bradford PA, Weiss WJ, Murphy TM, Sum PE, Projan SJ: In vitro and in vivo activities of tigecycline (GAR936), daptomycin, and comparative antimicrobial agents against glycopeptide-intermediate Staphylococcus aureus and other resistant Gram-positive pathogens. Antimicrob Agents Chemother 2002, 46:2595-2601.
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46. Ruzin A, Keeney D, Bradford PA: AcrAB efflux pump plays a role in decreased susceptibility to tigecycline in Morganella morganii. Antimicrob Agents Chemother 2005, 49:791-793. 47. Wenzel R, Bate G, Kirkpatrick P: Fresh from the pipeline: tigecycline. Nat Rev Drug Discov 2005, 4:809-810. This paper provides a nice update on clinical data and indications. It also gives an updated market analysis for intravenous antibiotics.
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