science/technology
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The specter of antibiotic resistance has roused pharmaceutical and biotechnology companies to seek new weapons to battle infections A currently available attack only a handful several years. The enterococci pick up of targets. Effectively conquering antibi- and exchange resistance genes quite easotic resistance will require expanding the ily, and they are already leading causes of ver since penicillin was launched available targets—some drugs that act on hospital infections. These strains have as a product in the early 1940s, the new targets are already in development. been able to transfer their resistance to specter of antibiotic resistance has In addition, biotechnology companies Staphylococcus aureus in the laboratory, threatened. Only recently, however, has that focus on antimicrobial research have and the fear is that they will eventually do resistance reached the point that some developed various assays to probe the so in nature as well. S. aureus is a particuinfections evade all available antibiot- bacterial genome to seek out more larly virulent organism that causes a broad array of problems, ranging from ics—even vancomycin, the glycopep- targets. tide that is often the "antibiotic of last Strains of the common intestinal bac- the mundane—such as pimples—to the resort." teria Enterococcus faecalis and E. faeciumserious and even life-threatening— For several years, many large phar- have been resistant to vancomycin for including pneumonia, osteomyelitis (inflammation of bone and bone marmaceutical companies lost interest row), endocarditis (heart valve infecin developing antibiotics, preferring tion), and bacteremia (a systemic to focus on chronic diseases. But the blood infection). increasing incidence of antibiotic resistance has brought a new sense There already are strains of glyof urgency to the discovery and decopeptide-intermediate S. aureus velopment of antibacterial drugs. At (GISA, sometimes called vancomyleast two new drugs are awaiting cin-intermediate S. aureus) with reapproval from the Food & Drug duced susceptibility to vancomycin. Administration. One of these—lineThefirstGISA strains were reported zolid from Pharmacia & Upjohn, in 1996 in Japan. In January, the Peapack, N.J.—is being touted as Centers for Disease Control & Prethe first drug in a completely new vention, Atlanta, announced that the class of antibiotics in many years. fourth confirmed case of GISA infecOther new antibacterial drugs are in tion in the U.S. had occurred in Illidevelopment. nois in April 1999. Thus far, GISA strains have been susceptible to oth"It's hard to get away from the er antibiotics. fact that antibiotic resistance is inevitable," says Julian E. Davies, an The most worrisome bacteria are emeritus microbiology professor at the methicillin-resistant S. aureus. the University of British Columbia, These bacteria are resistant to all anVancouver. "No matter what we are tibiotics except vancomycin. If they doing in terms of antibiotic discovery develop or acquire vancomycin reand antibiotic use, we have to realize sistance as well, medical doctors will that there's afinitelife for every antihave nothing in their arsenal to fight false color transmission electron micrograph biotic in terms of being able to use it Ashows them with. the effect of an antibiotic on Staphylococcus without fear of resistance. You can't aureus. At top Is an unaffected bacterium in the Bacteria are divided into two mastop it, but you can delay it." jor classes known as gram positive process of dividing. At bottom is the remains of a The antibacterial drugs that are bacterium destroyed by lysis. and gram negative, based on the reCelia M. Henry C&EN Washington
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science/technology ated [J. Am. Chem. Soc, 121, 1 1 9 2 2 (1999)]. Many academic researchers are trying noglycoside that is a substrate for The group evaluated the effectiveness to understand and find ways to thwart the resistance enzyme, but yields a of the modified aminoglycoside in kill antibiotic resistance mechanisms. One chemically unstable molecule upon ing both susceptible and resistant Es of them is Shahriar Mobashery, a phosphorylation. The p h o s p h a t e cherichia coll. When resistant E. coH chemistry professor at Wayne State group is eliminated spontaneously is treated with kanamycin, the mini University, Detroit. and the original antibiotic is regener- mum inhibitory concentration (MIC, One common way the amount of com that bacteria defeat ^^^^~~——^™ pound required to antibiotics is by us kill the bacteria) Antibiotic regenerates itself after being ing resistance en shoots up 500- to zymes that modify 1,000-fold, Moba phosphorylated by resistance enzyme the antibiotic and shery says. With the render it inactive. regenerating amino NHo For example, one glycoside, MIC in APH(3') = resistance enzyme widespread mecha creased only four ADP = adenosine diphosphate HO H9N nism for evading ATP = adenosine triphosphate fold. However, MIC aminoglycoside anti for the nonresistant biotics such as kanaE. coli was higher mycin is the phos for the regenerat ?OH phorylation of the ing aminoglycoside O-V^—OH antibiotic at the 3'than for kanamycin. hydroxyl position, "I don't think this which interferes specific drug will be with the interaction the replacement for of the drug and its kanamycin because target. (Aminogly its MIC for the noncosides inhibit pro resistant organism tein synthesis by has gone up a lit . Ρ u n ' \ NH2 0/\\ binding to the bac tle bit," Mobashery Ο "HO terial ribosome.) s a y s . "We have ?OH 9 OH Unstable Hydrated form demonstrated that Mobashery and phosphorylated form this strategy is per c o w o r k e r s have O^V^-OH fectly viable in low developed a self^OH ^OH ering MIC [for reregenerating ami
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sponse of the bacteria cell wall to a stain ing procedure developed in 1884 by the Danish physician Hans C. J. Gram. Grampositive bacteria retain the dye Gentian violet used in the staining process, whereas gram-negative bacteria do not. The cell walls of both types of bacte ria are made of peptidoglycan, which is composed of covalently linked poly saccharide and polypeptide chains. In gram-positive bacteria, a thick layer of peptidoglycan surrounds the plasma membrane. Gram-negative bacteria have a thin cell wall separated from the plasma membrane by an aqueous compartment known as the periplasmic space; an addi tional thick outer membrane coats the cell wall. The outer membrane consists of lipopolysaccharides, proteins, and phos pholipids, and accounts for the reason that many antibacterial drugs effective against gram-positive bacteria are inef fective against gram-negative bacteria. Antibacterial agents that are active against both classes of bacteria are deemed broad spectrum. Typical gram-positive bacteria in clude S. aureus, Streptococcus pneumon iae (the major cause of community42
MARCH 6, 2000 C&EN
acquired pneumonia—that is, pneumo nia contracted outside a hospital or institutional setting), and the enterococci. The roll call of gram-negative bacte ria includes Neisseria meningitidis (a cause of meningitis and the rarer meningococcemia), Escherichia coli, and Haemophilus influenzae. The current emphasis in antibacteri al drug development is on gram-positive infections because that's where the most immediate problems are. Ignoring the gram-negative bacteria now could mean trouble later, however. Alexander Rakowsky, a medical team leader in the Division of Anti-infective Drug Products of FDA's Center for Drug Evaluation & Research, worries that "gram negatives may become resistant as we begin to tackle the gram positives." Resistance is cyclical, Rakowsky notes. "You get one class under control and the other one jumps to the front and becomes the bully. I guess I'm fearful about not having a whole bunch of ex perimental things for some of the highly resistant gram negatives. People tend to go where there's a need at the time. The major need as far as resistance is con
cerned now is primarily in gram posi tives, but gram negatives will probably strike again."
Development of resistance Resistance takes several forms. The bacteria can alter the drug's target, pre vent the drug from reaching the target by mechanisms such as efflux pumps, or destroy or modify the drug so that it cannot bind to its target. Bacteria become resistant to antibiot ics either through genetic mutations or by acquiring resistance genes from oth er bacteria, which can trade genetic ma terial on pieces of DNA called plasmids. According to Davies, there is evidence that a combination of the two methods of acquiring resistance actually occurs. 'When an organism first meets an antibiotic, it appears that the first thing it will do is to mutate to get low-level resistance to the antibiotic," Davies explains. "At least it has, in a sense, breathing space." The bacteria can then achieve higher levels of resistance through a combination of further genet ic mutation and gene transfer between bacteria species.
Light triggers antibiotic's self-destruction
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A number of practices, lumped to gether under the heading "imprudent use," have contributed to the increase in antibiotic resistance. For example, anti biotics are prescribed for viral infec tions, even though they don't kill virus es. In addition, patients often fail to take all the antibiotic that's been prescribed because they feel better after one or two days. Both of these practices kill the most susceptible bacteria while allow ing the more resistant bacteria to sur vive. With less competition, the resis tant bacteria find it easier to thrive, a process known as selective pressure. Antibiotic resistance has "hit the in dustrialized world in a fashion that most did not expect," says Stuart B. Levy, a professor of medicine and of molecular biology and microbiology at Tufts Univer sity School of Medicine, Boston. Levy also is the founder and president of the Association for the Prudent Use of Antibi otics, which was established in 1981. "We have unbeatable organisms here, whereas when the organization was start ed, the major life-threatening problems were in the developing countries." Levy attributes the problems in developing na
sistant organisms]. It is a perfectly viable strategy to counter the cases of re sistance when the group is transferred to the drug itself." In another recently pub lished paper, Mobashery and coworkers described an environmentally friendly antibiotic that "self-de structs" after it has been eliminated from the body [J. Med. Chem., 43, 128 (2000)]. "One of the basic properties of antibiotics is that they don't readily get metabolized in the body/' Mobashery says. "They go through the body and are excreted in the fully active form." Once the antibiotic is in the environment, it applies selective evolutionary pres sure on the bacteria, allow ing them to develop resis tance. "It would be desir able after the antibiotic goes through the body if it could actually destroy itself. Then if s not a freestanding antibi otic in the environment that could select for resistance," Mobashery asserts. Mobashery and cowork
tions to poor sanitation and over-thecounter availability of antibiotics. However, Levy notes, the developing world's head start in drug resistance doesn't mean that the industrialized world hasn't caught up. 'We stockpile antibiotics in our homes. We demand them. We give them to our friends and neighbors. We overuse them and we misuse them." Levy agrees that antibiotic resistance within settings that are selective for the resistance is inevitable. He does not be lieve, however, that antibiotic resistance as a clinical problem is equally inevita ble. For antibiotic resistance to become a clinical problem to the extent that 5 to 10% of patients have resistant infections "takes a lot of antibiotic misuse and overuse," he says. Citing resistant S. pneumoniae (also known as pneumococcus) as an exam ple, Levy asks: "Why do you catch a re sistant pneumococcus? Because it's be ing confronted with antibiotics on a reg ular daily, hourly basis. We could isolate that as a clinical phenomenon, but it doesn't have to be a clinical problem. That is not inevitable. That's a question of how we use the drug."
ers synthesized an antibiotic that is an analog of cephalosporanic acid. Their antibiotic is modified at the 0 7 β posi tion with a hydrazine function protected by o-nitrobenzylcarbamate. The protect ing group can be removed by exposure to UV-visible light for several hours, as it might be in the environment The un protected hydrazine can then react intramolecularly with the lactam carbonyl, destroying the lactam group and elimi nating the antibacterial activity. Mobashery says that other destruc tive mechanisms could be used as well. For example, an antibiotic could be made pH sensitive so it would have limited survivability in aqueous solu tions. The best mechanism will depend on the conditions under which the drug is given and the form in which the drug is released. "If you want it to be destroyed in the sewer where there is no light, then you have to pursue a different strategy," Mo bashery says. "Should we have a step where strong light is shone at the sewer? These are not things that are in place right now because nobody has thought about this. Pm proposing in the literature for the first time that we do something about the 50 million lb per year of antibi otics that we are pumping into the envi ronment We should start thinking and rethinking how this could be changed and how it could be different than what it is right now."
Large quantities of antibiotics—pos sibly as much as 50% of the total pro duced—are used for purposes other than human therapy. They are fed to animals and fish and sprayed on fruit trees. Many of these antibiotics are used to prevent dis ease and promote growth at doses lower than would be used to treat infections. Some people worry that such use could contribute to antibiotic-resistant infections in humans, either through foodborne ill nesses caused by resistant bacteria or by the migration of resistance mechanisms between animal and human pathogens. A push to save essential antibiotics for human use is under way. For instance, Rep. Sherrod Brown (D-Ohio) intro duced a bill in the House of Representa tives in November 1999 that would pro hibit the use of essential antibiotics in livestock "unless there is a reasonable certainty of no harm to human health."
New drugs One new class of antibiotics is the oxazolidinones. Linezolid, developed by Pharmacia & Upjohn, is the first mem ber of the class to be filed with FDA and currently is awaiting approval. MARCH 6, 2000 C&EN 4 3
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science/technology gram-positive cytoplasmic Oxazolidinones have a five-membered membranes), and the bac heterocyclic ring that contains both nitro terial membrane potential. gen and oxygen. The drugs appear to The mechanism is not unwork by binding to one of the subunits of CH3SO3H derstood in detail, but the bacterial ribosome, the site of protein Francis P. Tally, executive synthesis. Linezolid prevents the interac vice president for scientif tion of the 30S ribosome subunit, mes ic affairs at Cubist, says senger RNA, and formyl methionine, Gemifloxacin that daptomycin is binding shutting down protein synthesis before it to an as-yet-unidentified starts. The drug is active against grampositive bacteria, including those that are called gemifloxacin, which inhibits type II molecule on the surface of gram-posi resistant to other drugs. topoisomerase, also known as DNA gy- tive organisms. The drug is assumed Even though linezolid hasn't even rase. This enzyme catalyzes the conver not to act on the cell wall because it is been approved yet, some bacteria over sion of relaxed DNA to a supercoiled active against a type of organism known time have shown signs of resistance to form. The new drug differs from other as L-form, which lack a cell wall. the drug, according to Wesley Mark fluoroquinolones by the presence of an Lilly discontinued development of Todd, therapeutic area clinical director at oxime-substituted aminomethyl pyrroli daptomycin because of perceived safety Pharmacia & Upjohn. These instances dine substituent at the C-7 position. Ac problems, Tally says. However, Cubist cording to Clarence L Young, group di has done additional clinical trials that in rector for anti-infectives clinical research dicate the safety of high doses of dapto and development and medical affairs, mycin is improved by administering the Ο gemifloxacin has been shown to be very drug once a day. When high doses are di II χχ—NHCCHj potent against a number of bacteria that vided into several smaller doses, patients cause respiratory tract infections, includ can experience skeletal muscle weak ing S. pneumoniae and Κ influenzae. ness, a reversible adverse effect. Tally ex F Ο Cubist Pharmaceuticals, Cambridge plains that the side effect is related to the Linezolid Mass., is developing another com "trough" concentration of daptomycin. "When you give a single high dose, pound, daptomycin, a cyclic lipopeptide licensed from Eli Lilly. The compound you get a high peak and a low trough. have occurred primarily in the company's has a decanoyl side chain linked to the When you give multiple doses, you get a "compassionate use" clinical trial. Most N-terminal tryptophan of a 13-amino- lower peak but a higher trough. If you let patients in that trial were exposed to the acid peptide. The C-terminal residue, the trough level go down below a certain drug for four weeks or more, longer than kynurenine, is linked to the molecule level during the day, then you don't see the scenario for which Pharmacia & Up via an ester bond on the hydroxyl side this adverse event The muscle is able to john is seeking regulatory approval. adjust" He is quick to point out that the chain of threonine. "The patients in that study were Daptomycin has been shown to be damage is reversible and that it only oc quite ill, and they had usually been ex bactericidal against vancomycin-resistant curs in skeletal muscle, not heart muscle. Xoma Corp., Berkeley, Calif., is de posed to multiple antibiotics before re enterococci, methicillin-resistant staphy ceiving the linezolid," Todd says. "In lococci (both S. aureus and S. epidermi-veloping a number of antibacterial prod many cases, they had areas where or dis), and S. aureus with reduced suscepti ucts based on bactericidal/permeabilityganisms could be harbored, and it was bility to vancomycin. Daptomycin dis increasing protein (BPI), which is found difficult to get blood or the drug into rupts multiple aspects of the bacterial in neutrophils (a type of white blood cell those areas." For example, he says, pa cytoplasmic membrane's function, in that ingests and kills bacteria). According tients had implanted prosthetic devices cluding peptidoglycan synthesis, lipotei- to Patrick J. Scannon, chief scientific and or isolated abscesses. In those cases it choic acid synthesis (a component of medical officer at Xoma, there are two is difficult to get adequate antibiotic in to kill the organism. "It's a situation where resistance is likely to occur for any antibiotic," Todd notes. Pharmacia & Upjohn is also looking for other members of the oxazolidinone class to increase the spectrum of activi ty. "If we were able to synthesize com pounds that affected some gram-nega u H tive organisms in addition to the gram Η Τ Π positives or killed the gram positives ΝΗ more quickly, those would be benefi cial," Todd says. HOC^L HOC Another new antibiotic is being devel 0 oped by SmithKline Beecham, King of Prussia, Pa. In December, SmithKline CH 3 Ο Daptomycin filed a new-drug application with FDA for its next-generation fluoroquinolone,
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