Reversing resistance: The next generation antibacterials.

Irrational antibiotic usage has led to vast spread resistance to available antibiotics, but we refuse to slide back to "preantibiotic era." The threat is serious with the "Enterococcus, Staphylococcous, Klebsiella, Acinetobacter, Pseudomonas and Enterobacter" organisms causing nosocomial infections that are difficult to treat because of the production of extended spectrum β-lactamases, carbapenamases and metallo-β-lactamases. Facing us is a situation where soon multidrug resistance would have spread across the globe with no antibiotics to withstand it. The infectious disease society of America and Food and Drug Administration have taken initiatives like the 10 × '20 where they plan to develop 10 new antibiotics by the year 2020. Existing classes of antibiotics against resistant bacteria include the carbapenems, oxazolidinones, glycopeptides, monobactams, streptogramins and daptomycin. Newer drugs in existing classes of antibiotics such as cephalosporins, aminoglycosides, tetracyclines, glycopeptides and β-lactamase inhibitors continue to get synthesized. The situation demands newer targets against bacterial machinery. Some of them include the peptidoglycantransferase, outer membrane protein of Pseudomonas, tRNA synthase, fatty acid synthase and mycobacterial ATP synthase. To curb the irrational and excessive usage of presently available antibiotics should be a priority if they are still to be kept in usage for the future.

Introduction

It had been proven in 1955 that resistance to antibiotics came up spontaneously by mutations even before antibiotics were discovered.[1] The bacteria harboring the genes responsible for resistance get selected each time we take an antibiotic, and such mutant bacteria possessing such genes have increased in prevalence with the increasing use of antibiotics. As can be predicted by this statement, resistance to all available drugs has progressively developed, only a bit early than expected. The success made initially in discovering new antibiotics and in treating infectious diseases led us to believe that infectious diseases could soon be eradicated. At present, we stand at a threat of going back to the preantibiotic era as many pathogens now possess resistance to almost all the antibiotics available. Until date, around 150 antibiotics have been approved [Figure 1], the last systemic antibiotic approved being ceftolozane in 2014, and last locally acting being finafloxacin in 2014.

Figure 1

Timeline of discovery of antibiotics

In this review, newer antibiotics from existing classes and also those directed against newer microbial targets are discussed according to their site of action. The information about them was taken from PubMed initially from review articles and specific original research articles were then sought to gain further information about each drug. To know the status of new drugs in the approval process clinicaltrials.gov and respective company's websites were referred.

Inhibitors of Cell-wall Synthesis

Peptidoglycan cell wall synthesis has been a popular target as it is vital for the bacteria, providing rigidity by virtue of a highly cross-linked lattice structure. Synthesis of peptidoglycan involves around 30 enzymes.[2] It is a polymer of a heterodimer. One of the monomer is acetyl-muramic acid linked to a pentapeptide chain (L-alanine-D-glutamate-L-lysine-D-alanine-D-alanine). The other sugar is acetyl-glucosamine. A protein with enzymatic property, penicillin binding protein-2 (PBP-2) catalyzes 2 steps: Glucosyltransferase action where these two sugars are linked together and dimers thus formed are then polymerized into long chains and transpeptidase reaction, which cross-bridges the pentapeptides attached to acetyl-muramic acid of two adjacent polymers by another penta-glycine bridge [Figure 2].

Figure 2

The structure of the peptidoglycan cell-wall

Penicillins and cephalosporins bind to the PBP-2 and inhibit transpeptidase activity thus preventing cross linking of the polymers, causing disruption of the cell wall and cell lysis. Resistance develops due to alteration of PBPs due to exchange of genes coding for PBPs between bacteria. Penicillins cannot bind to the altered PBPs, e.g., methicillin resistant Staphylococcus aureus (MRSA). Gram-negative organisms are less susceptible to penicillins as they have an outer membrane, which is difficult to cross for the penicillin molecule. However antibiotics like ampicillin and cephalosporins cross it by passing through “porins.” Pseudomonas is even difficult to kill as it lacks the usual high permeability porins. Other mechanisms of resistance include efflux pumps, β-lactamases and bio-films.

New drugs against resistant organisms are:

Glycopeptides

Vancomycin binds to D-ala-D-ala terminus of the nascent dimer, a site different from the PBP, preventing crosslinkage/transpeptidation. Its spectrum of activity includes MRSA and other Gram-positive organisms including Clostridium difficile. Resistance occurs by acquiring van-A plasmid, which substitutes D-ala-D-ala with D-ala-D-lactate. By 2002, MRSA had “updated” its status to vancomycin resistant S. aureus (VRSA) by acquiring the van-A plasmid from Enterococcus faecalis. Teicoplanin is a mixture of 6 closely related compounds. It is highly protein bound with a half-life of 100 h.[3] Like all below mentioned glycopeptides, teicoplanin is active against vancomycin resistant organisms. Telavancin is a derivative of vancomycin, approved in 2009. It has got an additional property of disrupting cell-membrane potential.[4] It has a concentration dependent bactericidal effect and is more potent than vancomycin. It is indicated for skin infections and for hospital acquired and ventilator associated pneumonias caused by MRSA and VRSA.[5] Oritavancin has a long half-life of 393 h[6] making stat-dose therapy possible, which is considerably convenient. It too has dual mechanism of action like telavancin. It is active not only against most of the resistant Gram-positive bacteria like MRSA, VRSA, but also against Bacillus anthracis and C. difficile.[78] As it is a semi-synthetic derivative of vancomycin, it too shares similar nephrotoxicity. The drug was finally acquired by “The Medicine Co” who have finished the recommended phase III studies after the Food and Drug Administration (FDA) declined to approve the drug due to lack of sufficient studies.[9] In February, 2014, NDA (new drug approval) was accepted by the FDA and finally approval was given in the same year.[10] Dalbavancin is a derivative of teicoplanin and it too has an additional mechanism of disrupting membrane potential of the bacteria. It is also highly protein bound and can therefore can be given once weekly, intravenously for 2 weeks.[6] It is active against MRSA, anerobic streptococci, claustridia and corynebacteria where the potency of dalbavancin was 4–32 times more than vancomycin, daptomycin or linezolid.[11] Phase III trials are over, Qualified Infectious Disease Product (QIDP) status has been given and a NDA filed in September, 2013, the drug got approved in 2014.[12]

Carbapenems

They also bind to the PBPs and inhibit cell wall synthesis. They are active against penicillin and cephalosporin resistant organisms. Resistance to carbapenems was first seen in enterobacteriaceae, which started producing Klebsiella pneumoniae carbapenemases (KPCs). Imipenem is now extensively used as a broad spectrum empiric therapy of nosocomial infections as it is active against both Gram-positive as well as Gram-negative organisms. The advantages of meropenem over imipenem are that it is not rapidly hydrolyzed by the renal dihydropeptidases and it has lesser propensity to cause seizures. Ertapenem was approved in 2001 for use in community acquired pneumonia (CAP), pelvic and urinary tract infections (UTIs).[13] Given as 1 g, once a day, its lack of activity against Pseudomonas excludes its use as an empirical agent for serious hospital acquired infections. Doripenem is active against resistant Pseudomonas.[2] It was developed in Japan and approved by the FDA in 2007. Doripenem has been observed to reduce the levels of valproic acid in epileptic patients and thus predispose them to seizures.[14] Faropenem, panipenem, biapenem are other carbapenems developed in Japan and used for more than a decade. Faropenem can be given orally and has been tested for acne,[15] sinusitis and pneumonia in clinical trials.

All carbapenems, though active against β-lactamases including extended spectrum β-lactamases (ESBLs), are inactive against MRSA.

Monobactams

The spectrum of action of aztreonam is limited to Gram-negative bacteria only. Due to structural dissimilarity, it can be given in penicillin allergic patients. Though it is active even in the presence of penicillinases and metallo-β-lactamases, it is not active against KPCs.[2]

Cephalosporins

Ceftaroline fosamil (a prodrug), which was developed by modifying the structure of the IV generation cephalosporin, cefozopran,[16] and ceftobiprole, both can be both considered as V generation cephalosporins. The route of administration is intravenous (i.v.). Ceftaroline has the ability to bind to the mutated PBP found in MRSA[17] and to the low affinity PBP5 of Enterococcus faecium.[18] Their spectrum of action is broad and includes MRSA, enterococci, pneumococci, anaerobes, VRSA and linezolid resistant bacteria but not against ESBL producing organisms[17] and Pseudomonas, but the combination of ceftaroline and avibactam expands its spectrum to resistant enterobacteriaceae also.[19] Common adverse drug reaction is Clostridium difficile associated diarrhea. Recent reports of hematologic toxicity and rash were reported in unexpectedly high rate of patients in a center.[20] In a recent trial, ceftobiprole was reported to be noninferior to ceftriaxone and linezolid in treatment of CAP.[21]

Fosfomycin

It inhibits phospho-enoyl-pyruvate transferase enzyme which is needed for the synthesis of acetyl-glucosamine, the building block for peptidoglycan.[22] It has been in use for more than 20 years for UTI as it is active against ESBL producing coliforms, MRSA and vancomycin resistant enterococci.

Peptidomimetics targeting Pseudomonas outer membrane protein

Many substances are secreted by lower as well as higher organisms as a natural defense against invading microbes. One among them is a peptide, protegrin-I, which apart from direct microbicidal activity have immunomodulatory action as well.[23] The outer membrane protein (Lpt D) of Pseudomonas and many other Gram-negative bacteria has the function of transporting lipopolysaccharide (LPS) to the outer leaflet of the outer membrane. Inhibiting the transport of LPS impairs the permeability barrier of the cell. Protegrin-I, a natural peptide binds to Lpt D and inhibits its function. Mimetics of this peptide have been developed which are specific only for Pseudomonas and inhibit its growth by a nonmembrane lytic mechanism of action.[24] POL 7080, an investigational compound gave good results in phase I studies in 2011.[25]

Glycosytransferase as a target

This enzyme is as vital as peptidyltransferase for the synthesis of cell wall. Glycosyltransferase is an attractive target due to the following reasons:

Inhibiting it will cause bactericidal action similar to that caused by penicillins

Function of glycosyltransferase is conserved among all bacteria and does not have any eukaryotic counterpart[26]

Technology has helped in complete understanding of the structure of the enzyme, its substrate and the reaction Therefore, synthesis of inhibitors is possible by predicting the structure activity relationship.

A natural product, moenomycin A [Figure 3], obtained from Streptomyces ghanaensis, is an antibiotic, which inhibits this enzyme and is more active against Gram-positive organisms much like the β-lactams. Interestingly no resistance was observed even after extensive usage in animal feeds as a growth promoting agent.[27]

Figure 3

The structure of moenomycin

The long lipid tail of moenomycin is responsible for many adverse effects and precludes use in humans.[28]

It has been shown now that the C, E and F trisaccharides are essential for activity.[29] Attaching a polypeptide is also necessary so as to resemble the natural substrate, acetyl-muramic acid with the pentapeptide chain attached to it. However, decreasing the lipid chain length leads to loss of activity. Efforts are being put and new molecules continue to be synthesized in the hope of utilizing this potential target, e.g., lipid II [Figure 4].

Figure 4

The structure of lipid II, which resembles a NAM-NAG dimer, along with the pentapeptide

Quinolones

Delafloxacin, in contrast to older quinolones has activity against Gram-positive organisms as well. Due to its anionic nature, it accumulates intracellularly and has a greater action at acidic pH,[30] making it a potential drug against S. aureus.[31] In a phase II trial comparing 300 mg twice a day i.v. delafloxacin with tigecycline, delafloxacin showed comparable efficacy (92%) and improved tolerability. In the study 70% of the S. aureus isolates were MRSA and 63% were levofloxacin resistant.[32] QIDP status has been given to delafloxacin by the FDA. Nemonoxacin is on the other hand a nonfluorinated quinolone, which also has higher efficacy as compared to levofloxacin against Gram-positive cocci like MRSA and vancomycin resistant Enterococcus (VRE).[33] Both these new quinolones are being developed as oral formulations and delafloxacin as an i.v. formulation as well. GSK’944 is a novel bacterial topoisomerase IIA inhibitor, which exhibits broad-spectrum activity. It has a different site of binding to the topoisomerase and DNA complex compared with other fluoroquinolones. At present, three phase I trials are being carried out. It has demonstrated activity against anthrax, plague and tularemia and is being developed in the U.S. under a public-private partnership to combat bio-terrorism.[34]

Inhibitors of RNA Synthesis

Fidaxomicin, approved in 2011, inhibits RNA polymerase of Gram-positive bacteria like C. difficile. It has a narrow spectrum of action and is not absorbed orally, hence is used to treat pseudomembranous colitis.

Ketolides

They bind to the 50S ribosomal RNA near peptidyltransferase site and block peptide chain elongation. Ketolides differ from macrolides by substitution of a 3-keto group for sugar moiety and this structural difference makes them less susceptible to the efflux mediated and methylase mediated mechanisms of macrolide resistance.[3] Ketolides are active against macrolide resistant strains. Telithromycin, a ketolide, approved in 2004 is not prescribed due to side effects like muscle weakness and hepatotoxicity.[35] Solithromycin is a novel fluoroketolide to be made available for both oral as well as i.v. administration. It is active against respiratory tract pathogens like pneumococcus, legionella, Moraxella and Haemophilus influenzae[3637] and is also proven to have an anti-inflammatory action as well.[38] Solithromycin, in a study on pregnant sheep, achieved good intrauterine concentrations,[39] thus may prove valuable in treating fetal/amniotic infections. It has been given QIDP status by the FDA and it entered phase 3 trials in 2013 for use against resistant pneumococci causing CAP.

Tetracyclines

They bind to 30S subunit and prevent binding of the incoming tRNA to the acceptor site. Resistance develops due to efflux pumps. Tigecycline is the first glycylcycline and is not a substrate for these efflux pumps and hence is active against tetracycline resistant bacteria. Bacterial resistance can also be due to production of a protein that displaces tetracycline binding to the 30S subunit. Tigecycline due to its different structure is resistant to this mechanism also. It has a broad spectrum of action, which includes Gram-positive as well as Gram-negative bacteria like MRSA, VRSA, enterobacteriacea, Acinetobacter, rickettsiae, chlamydia, legionella and rapidly growing mycobacteria. Proteus and Pseudomonas though are inherently resistant to all tetracyclines due to efflux pumps. Tigecycline is also available for i.v. use. Omadacycline is an aminomethylcycline and has a spectrum similar to tigecycline. It is active against bacteria resistant to tetracyclines as it is not a substrate for efflux pumps and neither is its action inhibited by the ribosomal protection protein.[40] QIDP status was given to this drug in January, 2013. Oral and i.v. formulations are currently in phase III. Eravacycline belongs to a category called fluorocyclines. In a phase II study, it was found to be as efficacious as ertapenem for the treatment of resistant Gram-negative organisms.[41] Its oral and parenteral formulations will enter phase III trials. Having both preparations makes i.v. to oral step-down possible. QIDP status was given in 2013[42] and in April, 2014, fast track status was given.

Aminoglycosides

Resistance to aminoglycosides develops by enzymes that deactivate them or by alteration of the 30S ribosome with which they bind. This alteration is by a methyltransferase enzyme.[43] Plazomicin, a synthetic derivative of sisomicin is active against bacteria that produce deactivating enzymes, but not against methyltransferases. In vitro trials demonstrated its efficacy against enterobacteriaceae, which were resistant to other aminoglycosides[44] and also against Gram-positive organisms like MRSA.[45] In 2014 it entered phase III trials.

Oxazolidinones

Linezolid was developed in the 1980s. It acts via binding to the 23S subunit of 50S ribosome and inhibits formation of the initial complex of t-RNA, m-RNA and ribosome. Although resistance can develop by efflux pumps, its prevalence is seen mainly in Gram-negative bacteria. It has action against MRSA and most other Gram-positive organisms. It has been kept as a reserve drug for the treatment of tuberculosis. Tedizolid is a prodrug which can be given i.v. or orally once daily. It finished phase III studies in July, 2012. NDA was submitted in October, 2013[46] and was approved by FDA in 2014.[47] Radezolid is another new oxazolidinone to which QIDP status has been given. It has a chemical structure, which allows for better tissue penetration and hence is proposed to have a good action against intracellular pathogens.[48] Radezolid and tedizolid both are active against linezolid resistant bacteria and are more potent as they have an additional binding site on the 23S ribosomal subunit.[49] Unlike linezolid, they do not cause myelosuppression. All oxazolidinones have excellent activity in treating pneumonia caused by resistant bacteria.

Streptogramins

Already in use in France for more than 30 years, they are used in a combination of 70:30 ratio of dalfopristin and quinupristin. Dalfopristin binds to 50S ribosome and induces a conformational change in it, which enhances binding of quinupristin. Quinupristin, like macrolides, blocks initial attachment of t-RNA and elongation step of protein synthesis. The synergistic effect is bactericidal.

Flopristin and linopristin in a ratio of 30:70 are newer streptogramins, which can be given orally. They are being developed in France and completed phase II trials for the treatment of CAP in 2009. They have the potential to kill VRE and treat pneumonia due to MRSA.

Daptomycin

It was discovered more than 25 years ago and belongs to a class called lipopeptides. It has a long lipid tail by which it attaches to the bacterial cell membrane. After insertion, it causes pore formation by redirecting the location of proteins involved in synthesis of cell wall,[50] thus causing potassium loss, which lyses the cell by depolarization. It is not dependent on active bacterial multiplication for its action.[51] It is rapidly bactericidal. It cannot be used in treating pneumonia as it gets inactivated by the surfactant present in lungs. Its novel mechanism makes it active against VRSA. It has been associated with few cases of myopathy.[52] Surotomycin is a novel lipopeptide, currently in phase III trials against C. difficile.

New target – tRNA synthase

Aminoacyl tRNA synthase is the enzyme which charges a t-RNA with its specific amino acid in the presence of an ATP. The amino acid gets attached to the 3’ end of the t-RNA, which has adenosine in the end. This loaded aminoacyl t-RNA then gets attached to the ribosome for protein synthesis. GSK’052 is a boron containing drug that forms an adduct via the boron atom to the 3’ terminal of the t-RNA and inhibits protein synthesis.[53] In early studies, it showed activity against Gram-negative bacteria.[54] It had entered phase II trials in 2012, but further development has stopped.

New target – Peptidyl deformylase inhibitors

The first amino acid incorporated in protein synthesis is methionine in eukaryotes and formyl-methionine in prokaryotes. Peptidyl deformylase removes the formyl group from the methionine during peptide elongation in prokaryotes. GSK’322 is a novel peptide agent which inhibits this enzyme and has completed phase I and phase II trials.[55] It is bactericidal against common respiratory tract pathogens like Streptococcus pneumoniae, H. influenzae, Streptococcus pyogenes and S. aureus.[56] Actinonin obtained from Streptomyces species was decades ago, known to have this function.[57] The dire need for new antibiotics has made researchers search for analogues of actinonin.

Inhibition of Fatty Acid Synthesis

Fatty acid synthase (FAS) is a multienzyme complex whose substrates are acetyl-coA and NADPH. There are seven enzymes involved in both eukaryotes and prokaryotes in synthesis of fatty acids. Bacterial FAS II is different from human FAS I. One of the seven enzymes is enoyl-reductase (FabI). The experimental drug, AFN-1252 inhibits staphylococcal enoyl-reductase and inhibits fatty acid synthesis in it.[58] Oral preparation has completed phase I and II studies and now phase I studies of i.v. preparation are to be conducted. An interesting fact is that isoniazid inhibits mycobacterial FabI. FabI is the lone enzyme for catalyzing the fatty acid synthesis step in S. aureus, H. influenzae, Moraxella catarrhalis and Escherichia coli.[59]

Antimycobacterials

Bedaquiline was approved in 2012 for the treatment of multidrug-resistant tuberculosis. It inhibits mycobacterial ATP synthase, thus starving it of energy and is bactericidal against dormant as well as active bacteria. It is effective more against intracellular bacteria than extracellular ones. It is supposed to be used for 6 months and has some side-effects of concern like QT prolongation and hepatotoxicity and some like pancreatitis and rhabdomyolysis in animal studies.[60] Delamanid inhibits synthesis of mycolic acid. It completed phase II trials with 481 patients in 2012 with good results.[61] It got approved in Europe in May, 2014.[62] It is a promising drug expected to enter clinical use in the near future.

β-lactamases and their Inhibitors

Production of β-lactamases, which hydrolyze the β-lactam ring, which is essential for antimicrobial activity, remains the most common mechanism of resistance. The genes which confer ability to produce β-lactamases are transferred among the bacteria by plasmids via conjugation or by bacteriophages. The β-lactamases are classified according to the Ambler classification based on the amino acid sequence into the following categories:[2]

Class A: ESBLs, which confer resistance to penicillins, cephalosporins, carbapenems. Among the most worrisome are the KPCs

Class B: Zinc dependent, also called as metallo-β-lactamases. They destroy all β-lactamases except aztreonam, e.g., - VIM, IMP and NDM (Verona integron encoded metallo-β-lactamase, New Delhi metallo-β-lactamase)

Class C: Deactivate cephalosporins

Class D: Deactivate cloxacillin.

Classes A, C and D require serine in their structure, hence are called serine-β-lactamases.

Avibactam is a new, potent β-lactamase inhibitor, which binds to it covalently. It has a broader action against all class A and C β-lactamases. It is being developed in combination with imipenem, cefepime, ceftazidime and ceftaroline and studies demonstrated efficacy against ESBLs and KPCs producing Gram-negative bacteria.[63]

A list of antibiotics under development has been summarized in Table 1.

Table 1

Spectrum of newer antibiotics in development

Legislative and Regulatory Body's Role

The United States has taken up the challenge of solving the health problem related to antibiotic resistance. Apart from funding research to find new antibiotics, U.S.A. has also taken the following initiatives:

The 10 × ’20 initiative

The infectious disease society of America (IDSA) has launched this campaign calling for the development and regulatory approval of 10 novel, efficacious and safe systemic antibiotics by 2020. For this, IDSA has joined hands with the congress, FDA, Centers for Disease Control, NIH and other stakeholder groups.[64] Out of the 10, telavancin and ceftaroline seem to fulfill criteria for 2 of them till now.

Food and Drug Administration safety and innovation act, July, 2012

It was drafted to increase funding to the FDA by charging money to the industries whose products FDA reviews. The Generating Antibiotics Incentive Now act was incorporated into FDA safety and innovation act to address issues regarding antibiotic resistance.[65]

Additional 5 years have been added to the patent for antibacterials and antifungals that treat serious and life threatening conditions and earmarking them for priority review and accelerated approval after giving them QIDP status

Until now trials were confined to infections like intra-abdominal or acute bacterial skin and skin structure infections. According to the new guidance, any site of the body can be chosen to test antibiotics, which target a specific pathogen, e.g., MRSA

Currently, NDAs are required to have 1000 patients’ safety data. Now, evaluation in 300 patients is suggested

The draft also says that FDA is now open to the use of surrogate markers for accelerated approval of antibiotics

To the disappointment of industry, QT prolongation studies might still be needed which take 6–9 months and enroll 250 patients.

Why is Antibiotic Resistance on the Rise?

Antibiotics are often used when not indicated, sometimes due to a lack of confidence in the diagnosis and sometimes the patients getting the antibiotic directly from the pharmacists for ailments like common cold. Patients, not being educated enough, stop the treatment on feeling symptomatically better. Incorrect dose and using antibiotics longer than needed also promote antibiotic resistance. Today's jet age offers both, us and resistant bacteria, rapid travel from one place to another as exemplified by the spread of resistance by tsunami victims and the victims of the gulf war.[66] Furthermore, patients from the west who come to India for cheaper treatment options take resistant bacteria back with them for example the New Delhi metallo-β-lactamase producing strain.

“Antibiotic stewardship,” as it is called, is the coordinated effort that should be put in order to curb this rising problem. Using antibiotics only when indicated and educating patients about the importance of completing the course of antibiotic prescribed are some of the ways. New/reserve antibiotics should be brought into use only if resistance is demonstrated to existing antibiotics. “De-escalation” should be practiced,[66] which is the shifting from a broad spectrum, empirical, antibiotic regimen to a narrow spectrum, pathogen specific, antibiotic to serious patients in the Intensive Care Unit after obtaining a sensitivity report. Sometimes, keeping an antibiotic away from clinical practice for a few years may cause return of susceptibility as exemplified by the interesting return of chloroquine susceptible plasmodia in African country of Malawi.[67]

Conclusion

At present we face a shortage of antibiotics which can act against resistant organisms found mostly in nosocomial infections like MRSA, VRSA, enterococci and Pseudomonas. “Pan-resistant” strains have emerged due to the misuse of antibiotics. However, by employing initiatives like antibiotic stewardship, we can reverse resistance. New antibiotics continue to emerge just like emerging and re-emerging diseases. Newer and attractive targets continue to get discovered. Though it may take time for them to be available in developing countries, new antibiotics are in development and are promising.