The next generation of ophthalmic fluoroquinolones, aimed at treating antibiotic-resistant bacteria, is just around the corner. Levofloxacin (Quixin, Santen, Napa, Calif.) is currently available, while gatifloxacin (Allergan) and moxifloxacin (Alcon) will be here soon. They all provide increased coverage when compared to previous generations of drugs. Here’s a look at how they perform against common organisms, and how they overcome antibiotic resistance.

Gatifloxacin and Moxifloxacin
The fourth-generation fluoroquinolones, moxifloxacin and gatifloxacin, are products of extensive research, and are designed to avoid the development of bacterial resistance. They’re available currently as the systemic medications Avelox (moxifloxacin, Bayer) and Tequin (gatifloxacin, Bristol-Myers Squibb). 

 • Gatifloxacin. This agent is, structurally, an 8-methoxy molecule, which imparts a high bioavailability and a wide spectrum of activity. Typical gram-negative and positive bacteria, Mycoplasma, Legionella, Chlamydia species and the anaerobe Propionibacterium acnes are susceptible to it.¹

In a recent study, gatifloxacin administered orally had high vitreal permeability comparable to the concentrations of topically applied antibiotics. This indicates that it might be used for prophylaxis during intraocular surgeries and open-globe injuries. The 20 patients in this study were instructed to take two 400-mg tablets 12 hours apart before surgery. The researchers drew blood, aqueous and vitreous samples preop, and performed assays using high-performance liquid chromatography. Vitreous levels of gatifloxacin were 5.4 times the MIC, or minimum inhibitory concentration, a standard susceptibility grading for antibiotics to specific pathogens, for Staphylococcus epidermidis, a common conjunctivitis pathogen; 10.4 times the MIC for Staphylococcus aureus, and exceeded the MICs for P. acnes, Streptococcus species and Proteus mirabilis. Gatifloxacin didn’t achieve effective levels for Enterococcus or Pseudomonas, but these infrequently cause endophthalmitis.¹ 

A Pseudomonas infection, an entity that the new agents are designed to fight.

In vitro isolates of gram-positive bacteria from conjunctivitis patients were four to 16 times more susceptible to gatifloxacin than to older fluoroquinolones. S. epidermidis had MICs of 2.0 µg/ml for gatifloxacin, 8.0 µg/ml for levofloxacin and 32 µg/ml for ciprofloxacin. Streptococcus pneumoniae was more susceptible to gatifloxacin as well, with an MIC value of 0.25 µg/ml, versus 1.0 µg/ml for levofloxacin and ciprofloxacin.² Gatifloxacin was also superior to ciprofloxacin in preventing keratitis in a rabbit LASIK model.³ 

 • Moxifloxacin. This is also an 8-methoxy fluoroquinolone, but differs from gatifloxacin in its different amino side-chain, which confers hydrophobicity and reduces efflux of the antibiotic from the bacterial cell, one of the key resistance mechanisms evolved by bacteria.⁴

Moxifloxacin has been shown to be safe and effective for treating bacterial conjunctivitis. In the first study of moxifloxacin-treated bacterial conjunctivitis, it killed 100 percent of S. epidermidis, S. aureus, P. aeruginosa and Serratia marcescens isolates, some of which where resistant to other common antibiotics. Moxifloxacin ophthalmic solution demonstrated an overall 81-percent eradication rate in 53 patients treated b.i.d. for only three days.⁵ The second study of 544 bacterial conjunctivitis patients resulted in 82 percent microbial eradication and 83 percent clinical cure for moxifloxacin at the test-of-cure visit on the ninth day after administration. Moxifloxacin was also superior to placebo at the day-three visit, with cure rates of 27 percent and 15 percent, respectively.⁶ In four studies,  336 pediatric patients and 392 adults had no safety concerns or adverse events that were significantly different from placebo.⁷

Levofloxacin
This agent has antibiotic activity against both gram-positive and gram-negative bacteria, classifying it as a third-generation fluoroquinolone. This is an improvement over ciprofloxacin and ofloxacin, which generally have less gram-positive activity. In 2001, a study found gram-positive bacteria to be 98-percent susceptible to levofloxacin, versus 78 percent and 61 percent for ofloxacin and ciprofloxacin, respectively.⁸ 

Levofloxacin has a high bioavailability that’s important for achieving MIC in the tear film and penetrating the anterior chamber. Following a single dose, the drug concentration in human tears remained above the MIC concentration for up to six hours, and was higher than previously reported tear concentrations for ciprofloxacin, ofloxacin and norfloxacin.⁹ When administered topically before cataract surgery, levofloxacin 0.5% achieved concentrations higher than MIC (90) in the anterior chamber. Compared with ciprofloxacin, the aqueous absorption rate of levofloxacin was four to seven times higher.¹⁰ These properties of levofloxacin still make it an important agent to use.

Overcoming Resistance
Resistance evolves in bacteria through genetic mutation and is accelerated by misuse of antibiotics. The basic ways bacteria resist fluoroquinolones are decreased cell wall permeability, altered enzymes that block antibiotic complexing and the use of efflux pumps that remove antibiotic molecules from the cell.¹¹ A single mutation in the gyrA gene, that codes for the DNA gyrase enzyme, can give a bacterium a resistance to older fluoroquinolones. Since fourth-generation drugs target two enzymes, however, a double mutation would be required to create resistant bugs, which is far less likely.¹² As each generation of fluoroquinolones is developed, it must be able to kill bacteria resistant to the previous generations.

A recent in vitro study compared the potencies of the newer generation of fluoroquinolones to second and third-generation drugs using standard susceptibility tests.

Researchers determined MICs for ciprofloxacin, ofloxacin, levofloxacin, gatifloxacin and moxifloxacin to 93 bacterial endophthalmitis isolates. The results proved that ciprofloxacin and ofloxacin-resistant S. aureus isolates were statistically susceptible to moxifloxacin. Organisms such as coagulase-negative staphylococci and Streptococcus viridans that were resistant to ciprofloxacin and ofloxacin were susceptible to levofloxacin, gatifloxacin and moxifloxacin. Moxifloxacin was the most potent fluoroquinolone for gram-positive bacteria, while ciprofloxacin, moxifloxacin, gatifloxacin and levofloxacin were of equal potency against gram-negative bacteria. Compared to older antibiotics, fourth-generation fluoroquinolones killed bacteria resistant to older agents, were more potent against gram-positive bacteria and were just as potent against gram-negative strains.¹²

Keratitis isolates of S. Aureus or P. aeruginosa from 200 patients were tested for susceptibility to levofloxacin, ciprofloxacin and ofloxacin in a different study. The results were determined from MICs and time-kill studies showing killing rates and decreases in colony counts. S. aureus isolates that were fluoroquinolone-resistant had low susceptibilities to levofloxacin (22 percent), ofloxacin (10 percent) and ciprofloxacin (3 percent). Ciprofloxacin demonstrated the highest potency against resistant P. aeruginosa. For more susceptible bacterial isolates, the three fluoroquinolones had equal potencies.¹³ 

 • Cross-resistance. A serious concern in the area of fluoroquinolone resistance is that bacteria that develop resistance to ciprofloxacin and older drugs could carry over these mechanisms to defeat the newest antibiotics. This cross-resistance could undermine efforts to control both ocular and systemic infections.

In one study, researchers evaluated two ciprofloxacin-resistant strains of S. pneumoniae for their susceptibility to the new generation of antibiotics, including gatifloxacin, moxifloxacin and levofloxacin. Scientists designed these strains to have increased resistance to ciprofloxacin by altering special parts of the bacterial DNA, known as the quinolone resistance-determining regions (QRDRs).

A Brief History of Fluoroquinolones

Fluoroquinolones were the first class of man-made antibiotics. All fluoroquinolones share a common molecular core with attached chemical domains designed to bind to bacterial DNA and the bacterial enzymes DNA gyrase and topoisomerase IV. DNA gyrase uncoils the supercoiled bacterial genome, allowing other replication enzymes, such as DNA polymerase, to function. DNA gyrase consists of four subunits, encoded by the gyrA and gyrB genes. DNA topoisomerase IV detaches daughter chromosomes after the completion of DNA replication. Both enzymes are necessary parts of the DNA replication and cell division process. The fluoroquinolone molecule forms a complex with the DNA and enzymes, locking the DNA in a lethal broken state. Since these enzymes are conserved in the bacterial genome, fluoroquinolones are bactericidal to many different bacteria.1,2 In 1958, the molecule 7-chloroquinolone was found to have antibiotic properties and, in 1962, nalidixic acid, the precursor to fluoroquinolones, was developed. Its narrow antibacterial spectrum, short half-life and high protein binding limited its effectiveness, though. In 1978, however, norfloxacin, a new molecule that improved on these three factors, was developed, making it the first fluoroquinolone. Modifying certain chemical domains on the central structural core of the molecules allows researchers to create additional antibiotics with improved effectiveness. In this manner, ofloxacin and ciprofloxacin were developed and are considered to be the second generation of fluoroquinolones. They improved on the gram-negative activity of norfloxacin and its duration of action, and, as such, are still important systemic and topical antibiotics. 1. Dong Y, Zhae X, Domagala J, Drlica K.  Effect of fluoroquinolone Concentration on Selection of Resistant Mutants of Mycobacterium bovis BCG and staphylococcus aureus.  Antimicrobial Agents and Chemotherapy, July 1999, p. 1756-1758, Vol 43. No. 7. 2. Drlica K, Zhao X.  DNA Gyrase, topoisomerase IV, and the 4-Quinolones.  Microbiology and Molecular Biology Reviews, Sept. 1997, p 377-392.

As was expected, ciprofloxacin didn’t prove effective against either strain. Levofloxacin showed some action against one, but had little against the other. Moxifloxacin and gatifloxacin, though, killed nearly 100 percent of the bugs.

Since the multi-drug efflux pump contributes to the growing resistance to ciprofloxacin and levofloxacin,¹⁴ the fact that fourth-generation fluoroquinolones kill resistant organisms with such effectiveness is impressive. 

 • Toxicity. To study toxicity, three concentrations of moxifloxacin were administered topically to the eyes of monkeys in a three-month study. Over this time period, no changes were observed, either through slit lamp exam, ophthalmoscopy or intraocular pressure measurement.¹⁵ An in vitro study using human corneal stromal keratocyte cells and endothelial cells to test the toxicity of different fluoroquinolones refutes that finding.

The study shows that moxifloxacin, gatifloxacin and ciprofloxacin were cytotoxic to the cells at relevant MIC levels after 15 or 30 minutes of incubation, while levofloxacin was cytotoxic to both cell types at one hour. Levofloxacin was less cytotoxic than the other molecules.¹⁶ It’s not certain if this would have any effect on the clinical setting in which these antibiotics will be used. The more important factor for these medications is the MIC, so it’s important to focus on clinically relevant, in vivo studies.

With history as a prologue, however, antibiotic resistance will continue to develop, given widespread and inappropriate use of various agents in physicians’ offices. Looking into our crystal ball, then, we see a fifth-generation agent in our future. 

Dr. Abelson, an associate clinical professor of ophthalmology at Harvard Medical School and senior clinical scientist at Schepens Eye Research Institute, consults in ophthalmic pharmaceuticals. Mr. Hallas is a research associate at Ophthalmic Research Associates in North Andover.

1. Hariprasad SM, Mieler WF, Holz ER.  Vitreous penetration of orally administered gatifloxacin in humans.  Trans Am Ophthalmol Soc 2002;100:153-160.
2. Long M, Jensen HG, Allergan Gatifloxacin Study Group.  Ocular bacteria from conjunctivitis patients: susceptibility to gatifloxacin and older flouroquinolones. ARVO 2003; Abstract 2115.
3. Donnenfeld ED, Solomon R, Doshi S, Perry HD, Snyder RW, Jensen HG.  Prophylaxis of streptococcus pneumoniae keratitis with gatifloxacin in a rabbit LASIK model.  ARVO 2002; B404.
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13. Kowalski RP, Pandya AN, Karenchak LM, Romanowski EG, Husted RC, Ritterband DC, Shah MK, Gordon YJ.  An in vitro resistance study of levofloxacin, ciprofloxacin, and ofloxacin using keratitis isolates of S. aureus and P. aeruginosa.  Ophthalmology 2001;108:10:1826-9.
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15. Bergamini MVW, Heaton J, McGee D, Hackett R, Hiddemen J.  A three month topical ocular toxicity study of moxifloxacin ophthalmic solutions in cynomologus monkeys.  ARVO 2003;4457.
16. Skelnik DL, Clark LA, Bezwada P.  Effect of drug concentration and exposure time of levofloxacin, ofloxacin, ciprofloxacin, gatifloxacin and moxifloxacin on human corneal endothelial cells and keratocytes.  ARVO 2003; Abstract 4739.